Pleuromutilin derivatives for the treatment of diseases mediated by microbes

ABSTRACT

A compound of formula (I) wherein n is 0 to 4; m is 0 or 1 with the proviso that the sulphur atom and R 3  are in vicinal position (if m=0 then R 3  is in position 2′, and if m=1 then R 3  is on position 1′); R is ethyl or vinyl; R 1  is hydrogen or (C1-6)alkyl; R 2  is hydrogen or —(C 3-6 )cycloalkyl, or —unsubstituted (C 1-6 )alkyl, or —(C 1-6 )alkyl substituted by one or more of —hydroxy; preferably one or two, —methoxy, —halogen, —(C 3-6 )cycloalkyl, or R 1  and R 2  together with the nitrogen atom to which they are attached form a 5 to 7 membered heterocyclic ring containing at least 1 nitrogen atom or 1 nitrogen and 1 additional heteroatome e.g. selected from N or O, or R 1  is hydroxy and R 2  is formyl; R 3  is OH, OR 4 , a halogen atom, or —with the proviso that R 3  is bound to 2′ R 3  represents —O—(CH 2 )P—O— with p is 2 or 3; R 4  is unsubstituted (C 1-6 )alkyl or (C 3-6 )cycloalkyl.

The present invention relates to organic compounds, namelypleuromutilins.

Pleuromutilin, a compound of formula A

is a naturally occurring antibiotic, e.g. produced by the basidomycetesPleurotus mutilus and P. passeckerianus, see e.g. The Merck Index, 13thedition, item 7617. A number of further pleuromutilins having theprinciple ring structure of pleuromutilin and being substituted at thehydroxy group have been developed, e.g. as antimicrobials.

From WO 02/04414 A1 pleuromutilin derivatives, e.g.14-O-[(Aminocyclohexan-2-yl (and -3-yl)-sulfanyl)-acetyl]-mutilins; fromWO 07/014409 A1e.g. 14-O-[((Mono- ordialkylamino)-cycloalkylsulfanyl)-acetyl]-mutilins and from WO 07/000004A1 e.g. [((Acyl-hydroxy-amino)-cycloalkylsulfanyl)-acetyl]-mutilins, areknown.

We have now found pleuromutilins with interesting activity combined withan unexpected remarkable metabolic stability.

The pleuromutilin derivatives according to the invention are compoundsof formula (I)

-   wherein-   n is 0 to 4;-   m is 0 or 1 with the proviso that the sulphur atom and R₃ are in    vicinal position (if m=0 then R₃ is in position 2′, and if m=1 then    R₃ is on position 1′);-   R is ethyl or vinyl;-   R₁ is hydrogen or (C₁₋₆)alkyl,-   R₂ is hydrogen or    -   (C₃₋₆)cycloalkyl, or    -   unsubstituted (C₁₋₆)alkyl, or    -   (C₁₋₆)alkyl substituted by one or more of        -   hydroxy; preferably one or two,        -   methoxy,        -   halogen,        -   (C₃₋₆)cycloalkyl, or-   R₁ and R₂ together with the nitrogen atom to which they are attached    form a 5 to 7 membered heterocyclic ring containing at least 1    nitrogen atom or 1 nitrogen and 1 additional heteroatome e.g.    selected from N or O, or-   R₁ is hydroxy and R₂ is formyl;-   R₃ is OH, OR₄, a halogen atom, or    -   with the proviso that R₃ is bound to 2′ R₃ represents        —O—(CH₂)_(p)—O— with p is 2 or 3;-   R₄ is unsubstituted (C₁₋₆)alkyl or (C₃₋₆)cycloalkyl.

Preferred compounds of the present invention are compounds of formula(II)

-   wherein n, R, R₁, R₂ and R₃ are as defined above.

More preferred compounds of the present invention are compounds offormula (III)

-   wherein n, R, R₁ and R₂ are as defined above.

Most preferred compounds of the present invention are

-   -   a compound of formula (IV)

-   -   a compound of formula (V)

and

-   -   a compound of formula (VI)

wherein n, R₁ and R₂ are as defined above.

Particularly preferred is a compound selected from the group consistingof

-   14-O-{[(1R,2R,4R)-4-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin-   14-O-{[(1S,2S,4S)-4-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin-   14-O-{[(1R,2R,5S)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin-   14-O-{[(1S,2S,5R)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin-   14-O-{[(1R,2R,4S)-4-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1S,2S,4R) diastereomer thereof-   14-O-{[(1R,2R,5R)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin-   14-O-{[(1S,2S,5S)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin-   14-O-{[(1R,2R,3R)-3-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1S,2S,3S) diastereomer thereof-   14-O-{[(1R,2R,4R)-4-Diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1S,2S,4S) diastereomer thereof-   14-O-{[(1R,2R,4R)-4-Ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1S,2S,4R) diastereomer thereof-   14-O-{[(1R,2R,5S)-5-Ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1S,2S,5R) diastereomer thereof-   14-O-{[(1R,2R,5S)-5-Diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1S,2S,5R) diastereomer thereof-   14-O-{[(1R,2R,4S)-4-Diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1S,2S,4R) diastereomer thereof-   14-O-{[(1R,2R,5R)-5-Diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1S,2S,5S) diastereomer thereof-   14-O-{[(1R,2R,3R)-3-Ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1S,2S,3S) diastereomer thereof-   14-O-{[(1R,2R,3R)-3-Diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1S,2S,3S) diastereomer thereof-   14-O-{[(1R,2R,4S)-4-(Formyl-hydroxy-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1S,2S,4R) diastereomer thereof-   14-O-{[(1R,2R,5S)-5-(Formyl-hydroxy-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1S,2S,5R) diastereomer thereof-   14-O-{[(1R,2R,3R/S)-3-(Formyl-hydroxy-amino)-2-hydroxy-cyclohexylsulfatyl]-acetyl}-mutilin    and the (1S,2S,3R/S) diastereomer thereof-   14-O-{[(1R,2R,5S)-2-Hydroxy-5-methylamino-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1S,2S,5R) diastereomer thereof-   14-O-{[(1R,2R,5S)-5-Allylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1S,2S,5R) diastereomer thereof-   14-O-{[(1R,2R,5S)-2-Hydroxy-5-(2-methoxy-ethylamino)-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1S,2S,5R) diastereomer thereof-   14-O-{[(1R,2R,4R*)-2-Hydroxy-4-(2-hydroxy-ethylamino)-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1S,2S,4S*) diastereomer thereof-   14-O-{[(1R,2R,4R*)-4-Cyclohexylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1S,2S,4S*) diastereomer thereof-   14-O-{[(1R,2R,4R*)-4-Cyclopropylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1 S,2S,4S*) diastereomer thereof-   14-O-{[(1R,2R,5S*)-4-Cyclopropylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1 S,2S,5R*) diastereomer thereof-   14-O-{[(1R,2R,4S*)-4-Cyclopropylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1S,2S,4R*) diastereomer thereof-   14-O-{[(1R,2R,5R*)-2-Hydroxy-5-morpholin-4-yl-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1S,2S,5S*) diastereomer thereof-   14-O-{[(1R,2R,5S*)-2-Hydroxy-5-morpholin-4-yl-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1S,2S,5R*) diastereomer thereof-   14-O-{[(1R,2R,5S)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin    and the (1S,2S,5R) diastereomer thereof-   14-O-{[(1R,2R,5S)-5-Ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin    and the (1 S,2S,5R) diastereomer thereof-   14-O-{[(1R,2R,5R)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin    and the (1 S,2S,5S) diastereomer thereof-   14-O-{[(1R,2R)-4-Aminomethyl-2-hydroxy-cyclohexylsufanyl]-acetyl}-mutilin    and the (1S,2S) diastereomers thereof-   14-O-{[5-Amino-2-chloro-cyclohexylsulfanyl]-acetyl}-mutilin-   14-O-{[4-Amino-2-chloro-cyclohexylsulfanyl]-acetyl}-mutilin-   14-O-[(4-Amino-1-hydroxy-cyclohexylmethylsulfanyl)-acetyl]-mutilin-   14-O-{[(1R,2R)-2-Hydroxy-5-(3-methylamino-propyl)-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1S,2S) diastereomers thereof-   14-O-{[(1R,2R)-2-Hydroxy-4-(3-methylamino-propyl)-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1S,2S) diastereomers thereof-   14-O-{[(1R,2R)-5-(3-Amino-propyl)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1S,2S) diastereomers thereof-   14-O-{[(1R,2R)-4-(3-Amino-propyl)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin    and the (1S,2S) diastereomer thereof-   14-O-{[(6R,8R)-8-Amino-1,4-dioxa-spiro[4.5]dec-6-ylsulfanyl]-acetyl}-mutilin    and the (6S,8S) diastereomer thereof-   14-O-{[4-Amino-2-methoxy-cyclohexylsulfanyl]-acetyl}-mutilin-   and-   14-O-{[5-Amino-2-methoxy-cyclohexylsulfanyl]-acetyl}-mutilin.

A compound provided by the present invention is herein also designatedas “compound(s) of (according to) the present invention”. A compound ofthe present invention includes a compound in any form, e.g. in freeform, in the form of a salt, in the form of a solvate and in the fora ofa salt and a solvate.

According to another aspect, the present invention provides a compoundof the present invention in the form of a salt and/or solvate.

The salts include preferably pharmaceutically acceptable salts, althoughpharmaceutically unacceptable salts are included, e.g. forpreparation/isolation/purification purposes.

A salt of a compound of the present invention includes a base salt or anacid addition salt. Pharmaceutically acceptable base salts includeammonium salts such as trimethylammonium salt, alkali metal salts suchas those of sodium and potassium, alkaline earth metal salts such asthose of calcium and magnesium, and salts with organic bases, includingsalts of primary, secondary and tertiary amines, such as isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexyl amine andN-methyl-D-glucamine, preferably sodium salts. Acid addition saltsinclude salts of a compound of the present invention with an acid, e.g.hydrogen fumaric acid, fumaric acid, tartaric acid,ethane-1,2-disulphonic acid, maleic acid, naphthalin-1,5-sulphonic acid,acetic acid, maleic acid, succinic acid, salicylic acid, azelaic acid,2-[(2,6-dichlorophenyl)amino]bezene acetic acid, hydrochloric acid,deuterochloric acid, preferably hydrochloric acid.

A compound of the present invention in free form may be converted into acorresponding compound in the form of a salt, and vice versa. A compoundof the present invention in free form or in the form of a salt and/or inthe form of a solvate may be converted into a corresponding compound infree form or in the form of a salt in non-solvated form, and vice versa.

A compound of of the present invention may exist in the form of isomersand mixtures thereof, e.g. optical isomers, diastereoisomers, cis/transconformers. A compound of the present invention may e.g. containasymmetric carbon atoms and may thus exist in the form of enatiomers ordiastereoisomers and mixtures thereof, e.g. racemates or diastereomericmixtures. Any asymmetric carbon atom may be present in the (R)-, (S)- or(R,S)-configuration, preferably in the (R)- or (S)-configuration.

For example, in a compound of formula I the carbon atom of thecycloalkyl ring which is attached to (CH₂)_(m)S group, the carbon atomof the cycloalkyl ring which is attached to the R₃ group, and the carbonatom of the cycloalkyl ring to which the (CH₂)_(n)N(R₁R₂) group isattached, all are asymmetric carbon atoms. Substituents attached to suchasymmetric carbon atom may thus exist in (R) and (S) configuration,including mixtures thereof. For example, if in a compound of formula IR₂ is substituted alkyl and that substituent is attached to a carbonatom of the side chain of such alkyl, the carbon atom to which suchsubstituent is attached is an asymmetric carbon atom and suchsubstituent may be in the (R)- and (S)-configuration, including mixturesthereof.

The configuration of substituents attached to asymmetric carbon atoms ofthe mutilin-tricyclus is preferably the same as in naturalpleuromutilin.

Isomeric mixtures may be separated as appropriate, e.g. according, e.g.analogously, to a method as conventional, to obtain pure isomers. Thepresent invention includes a compound of the present invention in anyisomeric form and in any isomeric mixture. The present invention alsoincludes tautomers of a compound of the present invention, wheretautomers can exist.

The compounds of the present invention exhibit pharmacological activityand are therefore useful as pharmaceuticals.

For example, the compounds of the present invention show antimicrobial,e.g. antibacterial, activity against gram positive bacteria, such ascoagulase positive Staphylococci, e.g. Staphylococcus aureus, coagulasenegative Staphylococci, e.g. Staphylococcus epidermidis, Staphylococcushaemolyticus, and Streptococci, e.g. Streptococcus pyogenes,Streptococcus pneurnoniae, Enterococci, e.g. Enterococcus faecium andListeria monocytogenes and against gram negative bacteria such asMoraxella, e.g. Moraxella catarrhalis, and Haemophilus, e.g. Haemophilusinfluenzae, and Legionella, e.g. Legionella pneumophila, Neisseriaceae,e.g. Neisseria gonorrhoeae, as well as against Mycoplasms, Chlamydia andobligatory anaerobes, e.g. Bacteroides fragilis, Clostridium difficile,Fusobacterium spp., and Propionibacterium spp.

The in vitro activity against aerobic bacteria was determined by AgarDilution Test or Microdilution Test according to the Clinical andLaboratory Standards Institute (CLSI, former NCCLS) Document M7-A7 Vol.26, No. 2: “Methods for dilution Antimicrobial Susceptibility Tests forBacteria that Grow Aerobically—Approved Standard; Seventh Edition(2006)”; and the test against anaerobic bacteria was performed accordingto the Clinical and Laboratory Standards Institute (CLSI, former NCCLS),Document , M11-A6, Vol. 24, No. 2: “Methods for AntimicrobialSusceptibility Testing of Anaerobic Bacteria—Approved Standard; SixthEdition (2004)” and the in vivo activity was tested by the septicaemiamouse model against Staphylococcus aureus.

Compounds of the present invention are therefore suitable for thetreatment and prevention of diseases which are mediated by microbes,e.g. by bacteria. Diseases which may also be treated include e.g.diseases mediated by Helicobacter, such as Helicobacter pylori, anddiseases mediated by Mycobacterium tuberculosis. Diseases which may alsobe treated include in general inflammatory diseases, where microbes aremediating said inflammation, e.g. including acne.

In another aspect the present invention provides a compound of thepresent invention for use as a pharmaceutical, preferably as anantimicrobial, such as an antibiotic, e.g. and an anti-anaerobic.

In another aspect the present invention provides a compound of thepresent invention for use in acne treatment.

In a further aspect the present invention provides a compound of thepresent invention for use in the preparation of a medicament for thetreatment of diseases, mediated by microbes, such as bacterials, forexample

-   -   diseases mediated by bacteria, e.g. selected from Staphylococci,        Streptococci, Enterococci;    -   diseases mediated by bacteria, e.g. selected from Moraxella,        Haemophilus, Legionella, Neisseriaceae;    -   diseases mediated by Helicobacter;    -   diseases mediated by Mycobacterium tuberculosis;    -   e.g. diseases mediated by Mycoplasms, Chlamydia and obligatory        anaerobes; and for the treatment of acne.

In a further aspect the present invention provides a method of treatmentof diseases mediated by microbes which comprises administering to asubject in need of such treatment an effective amount of a compound ofthe present invention e.g. in the form of a pharmaceutical composition.

In a further aspect the present invention provides a method of treatmentof acne which comprises administering to a subject in need of suchtreatment an effective amount of a compound of the present inventione.g. in the form of a pharmaceutical composition.

Treatment includes treatment and prophylaxis.

For antimicrobial and acne treatment, the appropriate dosage will, ofcourse, vary depending upon, for example, the chemical nature and thepharmakokinetic data of a compound of the present invention employed,the individual host, the mode of administration and the nature andseverity of the conditions being treated. However, in general, forsatisfactory results in larger mammals, for example humans, an indicateddaily dosage is in the range from about 0.5 mg to 3 g of a compound ofthe present invention conveniently administered, for example, in divideddoses up to four times a day.

A compound of the present invention may be administered by anyconventional route, for example enterally, e.g. including nasal, buccal,rectal, oral administration; parenterally, e.g. including intravenous,intramuscular, subcutaneous administration; or topically, e.g. includingepicutaneous, intranasal, intratracheal administration, e.g. in form ofcoated or uncoated tablets, capsules, injectable solutions orsuspensions, e.g. in the form of ampoules, vials, in the form of creams,gels, pastes, inhaler powder, foams, tinctures, lip sticks, drops,sprays, or in the form of suppositories, e.g. in analogous manner tomacrolides, such as erythromycins, e.g. clarithromycin or azithromycin.

A compound of the present invention may be administered in the form of apharmaceutically acceptable salt, e.g. an acid addition salt or a baseaddition salt, e.g. a metal salt, or in free form, optionally in theform of a solvate. A compound of the present invention in the form of asalt exhibits the same order of activity as the compound in free form,optionally in the form of a solvate.

A compound of the present invention may be used for pharmaceuticaltreatment according to the present invention alone or in combinationwith one or more other pharmaceutically active agents. Such otherpharmaceutically active agents include e.g. other antibiotics andantiinflammatory agents, and, if a compound of the present invention isused in the treatment of acne, other pharmaceutically agents includefurthermore agents which are active against acne.

Combinations include fixed combinations, in which two or morepharmaceutically active agents are in the same formulation; kits, inwhich two or more pharmaceutically active agents in separateformulations are sold in the same package, e.g. with instruction forco-administration; and free combinations in which the pharmaceuticallyactive agents are packaged separately, but instruction for simultaneousor sequential administration are given.

In another aspect the present invention provides a pharmaceuticalcomposition comprising a compound of the present invention in free formor in the form of a pharmaceutically acceptable salt and/or in the formof a solvate in association with at least one pharmaceutical, excipient,e.g. carrier or diluent, e.g. including fillers, binders,disintegrators, flow conditioners, lubricants, sugars and sweeteners,fragrances, preservatives, stabilizers, wetting agents and/oremulsifiers, solubilizers, salts for regulating osmotic pressure and/orbuffers.

In another aspect the present invention provides a pharmaceuticalcomposition according to the present invention, further comprisinganother pharmaceutically active agent.

Such pharmaceutical compositions may be manufactured according, e.g.analogously, to a method as conventional, e.g. by mixing, granulating,coating, dissolving or lyophilizing processes. Unit dosage form maycontain, for example, from about 0.5 mg to about 2000 mg, such as 10 mgto about 500 mg.

The compounds of the present invention are additionally suitable asveterinary agents, e.g. veterinary active compounds, e.g. in theprophylaxis and in the treatment of microbial, e.g. bacterial diseases,in animals, such as fowl, pigs and calves, e.g., and for diluting fluidsfor artificial insemination and for egg-dipping techniques.

In another aspect the present invention provides a compound of thepresent invention for use as a veterinary agent.

In a further aspect the present invention provides a compound of thepresent invention for the preparation of a veterinary composition whichis useful as a veterinary agent.

in another aspect the present invention provides a veterinary method forthe prophylaxis and the treatment of microbial, e.g. bacterial diseaseswhich comprises administering to a subject in need of such treatment aneffective amount of a compound of the present invention, e.g. in thefowl of a veterinary composition.

Examples 1 to 37 following thereafter exhibit MICs≤2 μg/ml againstStaphylococcus aureus ATCC49951 and Streptococcus pneumoniae ATCC49619.

The metabolic stability for compounds of the present invention wasdetermined by using cryopreserved primary human hepatocytes. 1×10⁶cells/mL were incubated in the absence and the presence of 5 and 25μg/mL of the test compounds at 37° C., 5% CO₂ for 4 hours. To evaluatethe in vitro degradation under assay conditions, a sample of each testcompound was incubated also in the absence of hepatocytes. Theincubation was stopped by freezing the reaction mixture. Afterultrafiltration and washing of the filter with acetonitrile, the samplesolution was analyzed for parent compound disappearance or metaboliteappearance using LC/MS (ion trap). The metabolic stability valuecorresponds to the detected parent compound in % after incubation.

At the compounds of the present invention, the introduction of the R₃group, preferably a hydroxy group, in vicinal position to the sulphursubstituent attached to the cyclohexyl ring reveals unexpectedimprovements in metabolic stability of the microbiologically activecomponents. Parent compound or active metabolite were more stable afterincubation with primary human hepatocytes in comparison to derivativeswithout the R₃ group, preferably the hydroxy group, attached to thecyclohexyl moiety of the pleuromutilin side chain.

For example after 4 h incubation with human hepatocytes at a compoundconcentration of 5 μg/mL, for a mixture of14-O-{[(1R,2R,5S)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride and the (1S,2S,5R) diastereomer hydrochloridethereof—Example 2 of the present invention—66% of parent compounds werefound, whereas for mixture of14-O-{[(1R,3R)-3-Amino-cyclohexylsulfanyl]-acetyl}-mutilin hydrochlorideand the (1S,3S) diastereomer hydrochloride thereof—analogous derivativewithout hydroxyl group—only 24% of parent compounds could be detected.

EXAMPLES

The trivial name mutilin refers to the IUPAC systematic name(1S,2R,3S,4S,6R,7R,8R,14R)-3,6-dihydroxy-2,4,7,14-tetramethyl-4-vinyl-tricyclo[5.4.3.0^(1,8)]tetradecan-9-one.In the examples, pleuromutilin derivatives are numbered in analogy tothe mutilin numbering system described by H. Berner (Berner, H.; Schulz,G.; Schneider H. Tetrahedron 1980, 36, 1807-1811):

Pleuromutilin thiol and pleuromutilin tosylate are compounds offormulae:

Example1—14-O-{[(1R,2R,4R)-4-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,4S) diastereomer hydrochloride Step A1.14-O-{[(1R,2R,4R)-4-tert-Butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S)diastereomer and14-O-{[(1R,2R,5S)-5-tert-Butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer and14-O-{[(1R,2R,4S)-4-tert-Butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R)diastereomer

To a solution of 3,4-epoxycyclohexyl-carbamic acid tert-butyl ester(Gómez-Sánchez, E.; Marco-Contelles J. Tetrahedron 2005, 61, 1207-1219.)(4.27 g, 20 mmol) and pleuromutilin thiol (Nagarajan, R. Eli Lilly andCompany 1978, U.S. Pat. No. 4,130,709) (7.10 g, 18 mmol) in 200 ml oftetrahydrofuran was added aluminum oxide (40 g, Brockmann activity I,neutral) and the resulting mixture was stirred for 40 hours at roomtemperature. The suspension was filtered and concentrated under reducedpressure. The residue was subjected to chromatography (silica,cyclohexane/ethyl acetate=1/1) to yield14-O-{[(1R,2R,4R)-4-tert-butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S)diastereomer (a) (R_(f)=0.38, 1.34 g, 12%) as well as a mixture of14-O-{[(1R,2R,5S)-5-tert-butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer and14-O-{[(1R,2R,4S)-4-tert-butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R)diastereomer (b) (R_(f)=0.26, 2.81 g, 25%) as colorless amorphous foams.

(a): ¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.74 (d, 1H, NH, J=7Hz), 6.13 (dd, 1H, 19-H, J=11 Hz and 18 Hz), 5.54 (d, 1H, 14-H, J=8 Hz),5.05 (m, 2H, 20-H), 4.90 (d, 1H, 2′-OH, J=5 Hz), 4.48 (d, 1H, 11-OH, J=6Hz), 3.55-3.20 (m, 6H, 1′-H, 2′-H, 4′-H, 11-H, 22-H), 2.40 (bs, 1H,4-H), 1.36 (s, 3H, 15-CH₃), 1.35 (s, 9H, tert-butyl), 1.06 (s, 3H,18-CH₃), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.62 (d, 3H, 16-CH₃, J=7 Hz).MS-ESI (m/z): 630 (MNa⁺), 1237 (2MNa⁺).

(b): ¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.70 (d, 1H, NH, J=7Hz), 6.12 (dd, 1H, 19-H, J=11 Hz and 18 Hz), 5.34 (d, 1H, 14-H, J=8 Hz),5.05 (m, 2H, 20-H), 4.82, 4.78 (d, 1H, 2′-OH, J=4 Hz), 4.48 (d, 1H,11-OH, J=6 Hz), 3.55-3.20 (m, 5H, 2′-H, 4′/5′-H, 11-H, 22-H), 2.97 (m,1H, 1′-H), 2.40 (bs, 1H, 4-H), 1.35 (s, 12H, 15-CH₃, tert-butyl), 1.05(s, 3H, 18-CH₃), 0.82 (d, 3H, 17-CH₃, J=7 Hz), 0.62 (d, 3-H, 16-CH₃, J=7Hz). MS-ESI (m/z): 630 (MNa⁺), 1237 (2MNa⁺).

or Step A2.14-O-{[(1R,2R,4R)-4-tert-Butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S)diastereomer and14-O-{[(1R,2R,5S)-5-tert-Butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer and14-O-{[(1R,2R,4S)-4-tert-Butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R)diastereomer

To a solution of 3,4-epoxycyclohexyl-carbamic acid tert-butyl ester (10g, 47 mmol) and pleuromutilin thiol (16.6 g, 42 mmol) in 200 ml ofmethanol and 20 ml of dioxane was added 2N NaOH (21 ml, 42 mmol) and theresulting mixture was stirred for 16 hours at room temperature. Aftercompletion of the reaction the pH was set to 7 with diluted HCl and thereaction mixture was concentrated under reduced pressure. The residuewas diluted with water and brine and extracted three times with ethylacetate. The organic layers were dried over sodium sulfate and filtered.The filtrate was concentrated under reduced pressure and afterchromatography (silica, cyclohexane/ethyl acetate=1/1)14-O-{[(1R,2R,4R)-4-tert-butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S)diastereomer (R_(f)=0.40, 3.1 g, 12% yield) as well as a mixture of14-O-{[(1R,2R,5S)-5-tert-butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer and14-O-{[(1R,2R,4S)-4-tert-butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R)diastereomer (R_(f)=0.25, 6.35 g, 25%) were obtained as colorlessamorphous foams.

or Step A3.14-O-{[(1R,2R,4R)-4-tert-Butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S)diastereomer and14-O-{[(1R,2R,5S)-5-tert-Butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer

To a solution of Pleuromutilin thiol (9.25 g, 23.5 mmol) in 100 ml ofacetonitrile (dried over 4 Å molecular sieve) was added1,5-diazabicyclo[4.3.0]non-5-ene (DBN, 2.9 μl, 23.5 mmol) and after 1hour of stirring at room temperature under argon atmosphere the mixturewas charged with syn-3,4-epoxycyclohexyl-carbamic acid tert-butyl ester(4.17 g, 19.5 mmol) and stirred for further 16 hours at roomtemperature. The reaction mixture was concentrated under reducedpressure. The residue was charged with water and brine and extractedthree times with dichloromethane. The organic layers were dried oversodium sulphate and filtered. The filtrate was concentrated underreduced pressure and subjected to chromatography (silica,cyclohexane/ethyl acetate=1/1) to yield14-O-{[(1R,2R,4R)-4-tert-butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S)diastereomer (R_(f)=0.38, 5.07 g, 43%) as well as14-O-{[(1R,2R,5S)-5-tert-butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (R_(f)=0.25, 2.95 g, 16.5%) as colorless amorphous foams.

Step B.14-O-{[(1R,2R,4R)-4-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S)diastereomer

To a solution of14-O-{[(1R,2R,4R)-4-tert-butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S)diastereomer (1.34 g, 2.20 mmol) in 75 ml of dichloromethane was addedtrifluoroacetic acid (4 ml) at 4° C. and stirred for 5 hours at roomtemperature. The reaction mixture was diluted with dichloromethane andcautiously poured into a saturated NaHCO₃ solution. The phases wereseparated and the aqueous layer was washed two times withdichloromethane. The combined organic layers are dried over sodiumsulfate and filtered. After chromatography (silica, ethylacetate/methanol/35% ammonia solution=50/50/1)14-O-{[(1R,2R,4R)-4-amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S)diastereomer (745 mg, 67% yield) was obtained as colorless amorphousfoam.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.14 (dd, 1H, 19-H, J=11Hz and 18 Hz), 154 (d, 1H, 14-H, J=8 Hz), 5.05 (m, 2H, 20-H), 4.50 (d,1H, 11-OH, J=6 Hz), 3.50-3.20 (m, 5H, 2′-H, 4′-H, 11-H, 22-H), 2.55 (m,1H, 1′-H), 2.40 (bs, 1H, 4-H), 1.35 (s, 3H, 15-CH₃), 1.06 (s, 3H,18-CH₃), 0.82 (d, 3H, 17-CH₃, J=7 Hz), 0.62 (d, 3H, 16-CH₃, J=7 Hz).MS-ESI (m/z): 508 (MH⁺), 530 (MNa⁺), 1015 (2MH⁺), 1037 (2MNa⁺).

Step C.14-O-{[(1R,2R,4R)-4-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,4S) diastereomer hydrochloride

A solution of14-O-{[(1R,2R,4R)-4-amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S)diastereomer (325 mg, 0.64 mmol) in 20 ml of dioxane was treated with 1NHCl (0.64 ml, 0.64 mmol). After stirring at room temperature for 30minutes the solution was lyophilized to obtain14-O-{[(1R,2R,4R)-4-amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,4S) diastereomer hydrochloride (quantitative yield)as colorless amorphous solid.

¹H NMR (500 MHz, DMSO-d₆, δ, ppm, inter alia): 7.6 (bs, 3H, NH₃ ⁺), 6.14(dd, 1H, 19-H, J=11 Hz and 18 Hz), 5.55 (d, 1H, 14-H, J=8 Hz), 5.05 (m,2H, 20-H), 4.52 (d, 1H, 11-OH, J=6 Hz), 3.50-3.20 (m, 4H, 2′-H, 11-H,22-H), 3.03 (m, 1H, 4′-H), 2.53 (m, 1H, 1′-H), 2.40 (bs, 1H, 4-H), 1.37(s, 3H, 15-CH₃), 1.06 (s, 3H, 18-CH₃), 0.82 (d, 3H, 17-CH₃, J=7 Hz),0.62 (d, 3H, 16-CH₃, J=7 Hz). MS-ESI (m/z): 508 (MH⁺), 530 (MNa⁺), 1015(2MH⁺), 1037 (2MNa³⁰), 542 (MCl⁻).

Example1A—14-O-{[(1S,2S,4S)-4-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinand14-O-{[(1R,2R,4R)-4-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin

The mixture of14-O-{[(1R,2R,4R)-4-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S)diastereomer (12 g, 23.6 mmol) from Example 1 Step B was separated on achiral column (250×20 mm CHIRALCEL OD-H,n-heptane/ethanol/diethylamine=80/20/0.1) to yield14-O-{[(1S*,2S*,4S*)-4-amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin(a) (early eluting compound, 4.76 g, 37% yield, uncorrected) and14-O-{[(1R*,2R*,4R*)-4-amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin(b) (late eluting compound, 3.63 g, 30% yield, uncorrected) as colorlessamorphous foams.

(a): ¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.13 (dd, 1H, 19-H,J=11 Hz and 18 Hz), 5.54 (d, 1H, 14-H, J=8 Hz), 5.05 (m, 2H, 20-H), 4.50(d, 1H, 11-OH, J=6 Hz), 3.50-3.20 (m, 5H, 2′-H, 4′-H, 11-H, 22-H), 2.55(m, 1H, 1′-H), 2.40 (bs, 1H, 4-H), 1.35 (s, 3H, 15-CH₃), 1.05 (s, 3H,18-CH₃), 0.82 (d, 3H, 17-CH₃, J=7 Hz), 0.62 (d, 3H, 16-CH₃, J=7 Hz).MS-ESI (m/z): 508 (MH⁺), 530 (MNa⁺), 1015 (2MH⁺), 1037 (2MNa⁺), 506(M-H)⁻, 542 (MCl⁻).

(b): ¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.13 (dd, 1H, 19-H,J=11 Hz and 18 Hz), 5.54 (d, 1H, 14-H, J=8 Hz), 5.05 (m, 2H, 20-H), 4.50(d, 1H, 11-OH, J=6 Hz), 3.50-3.20 (m, 5H, 2′-H, 4′-H, 11-H, 22-H), 2.55(m, 1H, 1′-H), 2.40 (bs, 1H, 4-H), 1.35 (s, 3H, 15-CH₃), 1.05 (s, 3H,18-CH₃), 0.82 (d, 3H, 17-C₃, J=7 Hz), 0.62 (d, 3H, 16-CH₃, J=7 Hz).MS-ESI (m/z): 508 (MH⁺), 530 (MNa⁺), 1015 (2MH⁺), 1037 (2MNa⁺), 506(M-H)⁻, 542 (MCl⁻).

Example2—14-O-{[(1R,2R,5S)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,5R) diastereomer hydrochloride Step A.14-O-{[(1R,2R,5S)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer and14-O-{[(1R,2R,4S)-4-Amino-2-hydroxy-cyclhexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R)diastereomer

A mixture of14-O-{[(1R,2R,5S)-5-tert-butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer and14-O-{[(1R,2R,4S)-4-tert-butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R)diastereomer (1.12 g, 1.84 mmol) from Example 1 Step A was treatedaccording to the method of Example 1 Step B. After work up andchromatography of the reaction mixture (silica, ethylacetate/methanol/35% ammonia solution=50/50/1)14-O-{[(1R,2R,5S)-5-amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (a) (R_(f)=0.33, 524 mg, 56% yield) and14-O-{[(1R,2R,4S)-4-amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R)diastereomer (b) (R_(f)=0.22, 160 mg, 17%) were obtained as colorlessamorphous foams.

(a): ¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.13 (dd, 1H, 19-H,J=11 Hz and 18 Hz), 5.53 (d, 1H, 14-H, J=8 Hz), 5.05 (m, 2H, 20-H), 4.51(d, 1H, 11-OH, J=6 Hz), 3.48 (m, 1H, 2′-H), 3.42 (m, 1H, 11-H),AB-system (ν_(A)=3.37, ν_(B)=3.23, 22-H, J=19 Hz), 2.98 (m, 1H, 1′-H),2.82 (m, 1H, 5′-H), 2.40 (bs, 1H, 4-H), 1.36 (s, 3H, 15-CH₃), 1.06 (s,3H, 18-CH₃), 0.82 (d, 3H, 17-CH₃, J=7 Hz), 0.63 (d, 3H, 16-CH₃, J=7 Hz).

(b): ¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.14 (dd, 1H, 19-H,J=11 Hz and 18 Hz), 5.54 (d, 1H, 14-H, J=8 Hz), 5.06 (m, 2H, 20-H), 4.51(bs, 1H, 11-OH), 3.79 (m, 1H, 2′-H), 3.42 (m, 1H, 11-H), AB-system(ν_(A)=3.33, ν_(B)=3.23, 22-H, J=15 Hz), 3.04 (m, 1H, 4′-H), 2.82 (m,1H, 1′-H), 2.40 (bs, 1H, 4-H), 1.37 (s, 3H, 15-CH₃), 1.06 (s, 3H,18-CH₃), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.63 (d, 3H, 16-CH₃, J=7 Hz).

Step B.14-O-{[(1R,2R,5S)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,5R) diastereomer hydrochloride

14-O-{[(1R,2R,5S)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (516 mg, 1.02 mmol) was treated according to the method ofExample 1 Step C to obtain14-O-{[(1R,2R,5S)-5-amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,5R) diastereomer hydrochloride (533 mg, 96% yield)as colorless amorphous solid.

¹H NMR (500 MHz, DMSO-d₆, δ, ppm, inter alia): 7.7 (bs, 3H, NH₃ ⁺),6.13, 6.12 (2dd, 1H, 19-H, J=11 Hz and 18 Hz), 5.55 (d, 1H, 14-H, J=8Hz), 5.05 (m, 2H, 20-H), 4.53 (d, 1H, 11-OH, J=6 Hz), 3.70 (m, 1H,2′-H), 3.42 (t, 1H, 11-H, J=6 Hz), 3.35 (m, 2H, 22-H), 3.09 (m, 2H,1′-H, 5′-H), 2.40 (bs, 1H, 4-H), 1.35 (s, 3H, 15-CH₃), 1.05 (s, 3H,18-CH₃), 0.82 (d, 3H, 17-CH₃, J=7 Hz), 0.63 (d, 3H, 16-CH₃, J=7 Hz).MS-ESI (m/z): 508 (MH⁺), 530 (MNa⁺), 1015 (2MH⁺), 1037 (2MNa⁺), 542(MCl⁻).

Example2A—14-O-{[(1R,2R,5S)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinand14-O-{[(1S,2S,5R)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin

The mixture of14-O-{[(1R,2R,5S)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (4.91 g, 9.67 mmol) from Example 2 Step A was separated ona chiral column (250×20 mm CHIRALCEL OD-H,n-heptane/isopropanol/diethylamine=80/20/0.1) to yield14-O-{[(1R*,2R*,5S*)-5-amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin(a) (early eluting compound, 2.07 g, 42% yield, uncorrected) and14-O-{[(1S*,2S*,5R*)-5-amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin(b) (late eluting compound, 2.36 g, 48% yield, uncorrected) as colorlessamorphous foams.

(a): ¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.13 (dd, 1H, 19-H,J=11 Hz and 18 Hz), 5.53 (d, 1H, 14-H, J=8 Hz), 5.05 (m, 2H, 20-H), 4.72(d, 1H, 2′-OH, J=4 Hz), 4.50 (d, 1H, 11-OH, J=6 Hz), 3.47 (m, 1H, 2′-H),3.45-3.20 (m, 3H, 11-H, 22-H), 2.98 (m, 1H, 1′-H), 2.80 (m, 1H, 5′-H),2.40 (bs, 1H, 4-H), 1.36 (s, 3H, 15-CH₃), 1.06 (s, 3H, 18-CH₃), 0.81 (d,3H, 17-C₃, J=7 Hz), 0.63 (d, 3H, 16-CH₃, J=7 Hz). MS-ESI (m/z): 508(MH⁺), 530 (MNa⁺), 1015 (2MH⁺), 1037 (2MNa⁺), 506 (M-H)⁻, 542 (MCl⁻).

(b): ¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.13 (dd, 1H, 19-H,J=11 Hz and 18 Hz), 5.53 (d, 1H, 14-H, J=8 Hz), 5.05 (m, 2H, 20-H), 4.72(d, 1H, 2′-OH, J=4 Hz), 4.50 (d, 1H, 11-OH, J=6 Hz), 3.47 (m, 1H, 2′-H),3.45-3.20 (m, 3H, 11-H, 22-H), 2.98 (m, 1H, 1′-H), 2.80 (m, 1H, 5′-H),2.40 (bs, 1H, 4-H), 1.36 (s, 3H, 15-CH₃), 1.06 (s, 3H, 18-CH₃), 0.81 (d,3H, 17-CH₃, J=7 Hz), 0.63 (d, 3H, 16-CH₃, J=7 Hz). MS-ESI (m/z): 508(MH⁺), 530 (MNa⁺), 1015 (2MH⁺), 1037 (2MNa⁺), 506 (M-H)⁻, 542 (MCl⁻).

Example3—14-O-{[(1R,2R,4S)-4-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,4R) diastereomer hydrochloride

14-O-{[(1R,2R,4S)-4-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R)diastereomer (152 mg, 0.30 mmol) from Example 2 Step A was treatedaccording to the method of Example 1 Step C to obtain14-O-{[(1R,2R,4S)-4-amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,4R) diastereomer hydrochloride (148 mg, 91% yield)as colorless amorphous solid.

¹H NMR (500 MHz, DMSO-d₆, δ, ppm, inter alia): 7.8 (bs, 3H, NH₃ ⁺),6.14, 6.13 (2dd, 1H, 19-H, J=11 Hz and 18 Hz), 5.55 (d, 1H, 14-H, J=8Hz), 5.20 (d, 1H, 2′-OH), 5.05 (m, 2H, 20-H), 4.53 (d, 1H, 11-OH, J=6Hz), 3.88 (m, 1H, 2′-H), 3.42 (t, 1H, 11-H, J=6 Hz), 3.32 (m, 2H, 22-H),3.22 (m, 1H, 4′-H), 2.92 (m, 1H, 1′-H), 2.40 (bs, 1H, 4-H), 1.35 (s, 3H,15-CH₃), 1.05 (s, 3H, 18-CH₃), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.63 (d,3H, 16-CH₃, J=7 Hz). MS-ESI (m/z): 508 (MH⁺), 530 (MNa⁺), 1015 (2MH⁺),1037 (2MNa⁺), 542 (MCl⁻).

Example4—14-O-{[(1R,2R,5R)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5S)diastereomer Step A.tert-Butyl-dimethyl-(cis-3,4-epoxycyclohexyloxy)-silane

To a solution of 3-cyclohexen-1-ol (Amburgey, J. C.; Shuey, S. W.;Pedersen, L. G.; Hiskey R., Bioorganic Chemistry 1994, 22, 172-197.) (10g, 102 mmol) in 200 ml of dichloromethane was added vanadylacetylacetonate (0.5 g, cat.) and tert-butyl hydroperoxide (20.4 ml 5.5Min decane, 112 mmol) and stirred overnight at room temperature. Theresulting reaction mixture was treated with tert-butyldimethylsilylchloride (16.9 g, 112 mmol), imidazole (9.02 g, 132 mmol) and4-dimethylaminopyridine (2.49 g, 20 mmol) at 4° C. and stirred for 5hours at room temperature. The reaction mixture was diluted withdichloromethane and subsequently extracted with 10% NaHSO₃ solution,saturated NaHCO₃ solution and brine. The organic layer was dried oversodium sulfate and filtered. The filtrate was concentrated under reducedpressure and subjected to chromatography (silica, cyclohexane/ethylacetate=15/1) to yieldtert-butyl-dimethyl-(cis-3,4-epoxycyclohexyloxy)-silane (R_(f)=0.35,18.3 g, 79% yield) as colorless oil.

¹H NMR (500 MHz, DMSO-d₆, δ, ppm): 3.55 (m, 1H), 3.00 (m, 2H), 2.15 (m,1H), 2.00 (m, 1H), 1.80 (m, 1H), 1.50 (m, 1H), 1.35 (m, 1H), 1.35 (m,1H), 1.25 (m, 1H), 0.83 (s, 9H, tert-butyl), 0.0 (s, 9H, Si(CH₃)₂).

Step B.14-O-{[(1R,2R,5S)-5-(tert-Butyl-dimethyl-silyloxy)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer

tert-Butyl-dimethyl-(cis-3,4-epoxycyclohexyloxy)-silane (6.41 g, 28mmol) was treated with pleuromutilin thiol according to the method ofExample 1 Step A2. Crude14-O-{[(1R,2R,5S)-5-(tert-butyl-dimethyl-silyloxy)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer was obtained as colorless amorphous foam which was directlyused for the next step.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.13 (dd, 1H, 19-H, J=11Hz and 18 Hz), 5.52 (d, 1H, 14-H, J=8 Hz), 5.05 (m, 2H, 20-H), 4.78 (dd,1H, 2′-OH, J=5 Hz and 6 Hz), 4.48 (d, 1H, 11-OH, J=6 Hz), 3.88 (m, 1H,5′-H), 3.15-3.45 (m, 4H, 2′-H, 11-H, 22-CH2), 2.92 (m, 1H, 1′-H), 2.38(bs, 1H, 4H), 1.36 (s, 3H, 15-CH₃), 1.05 (s, 3H, 18-CH₃), 0.86 (s, 9H,tert-butyl), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.63 (d, 3H, 16-CH₃, J=7 Hz),0.0 (s, 6H, Si(CH₃)₂).

Step C.14-O-{[(1R,2R,5S)-2,5-Dihydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer

To a solution of14-O-{[(1R,2R,5S)-5-(tert-butyl-dimethyl-silyloxy)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (9.46 g, 15.1 mmol) in 25 ml of tetrahydrofuran a mixtureof acetic acid and water (3:1, 100 ml) was added and stirred for 2 daysat 40° C. The reaction mixture was concentrated nearly to dryness underreduced pressure and the residue was dissolved in ethyl acetate andsubmitted to chromatography (silica, cyclohexane/ethyl acetate=1/3) toyield the14-O-{[(1R,2R,5S)-2,5-dihydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (R_(f)=0.27, 7.07 g, 92% yield) as colorless amorphousfoam.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.12 (dd, 1H, 19-H, J=11Hz and 18 Hz), 5.53 (d, 1H, 14-H, J=8 Hz), 5.05 (m, 2H, 20-H), 4.72 (dd,1H, 2′-OH, J=2 Hz and 5 Hz), 4.48 (d, 1H, 11-OH, J=6 Hz), 4.43 (t, 1H,5′-OH), 3.68 (m, 1H, 5′-H), 3.45-3.20 (m, 4H, 2′-H, 11-H, 22-H), 2.94(m, 1H, 1′-H), 2.38 (bs, 1H, 4-H), 1.36 (s, 3H, 15-CH₃), 1.06 (s, 3H,18-CH₃), 0.82 (d, 3H, 17-CH₃, J=7 Hz), 0.63 (d, 3H, 16-CH₃, J=7 Hz).MS-ESI (m/z): 531 (MNa⁺), 1039 (2MNa⁺).

Step D.14-O-{[(1R,2R,5S)-2-Hydroxy-5-methanesulfonyloxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer

To a solution of14-O-{[(1R,2R,5S)-2,5-dihydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (6.07 g, 11.9 mmol) in 36 ml of pyridine was addedmethanesulfonyl chloride (1.1 ml, 14.3 mmol) and the resulting mixturewas stirred overnight at room temperature. Subsequently the solvent wasevaporated under reduced pressure; the residue was diluted with 1N HCland extracted three times with ethyl acetate. The combined organiclayers were washed with brine, dried over sodium sulfate and filtered.The filtrate was concentrated and purified by column chromatography(silica, cyclohexane/ethyl acetate=1/1) to yield14-O-{[(1R,2R,5S)-2-hydroxy-5-methanesulfonyloxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (R_(f)=0.15, 2.55 g, 36% yield) as colorless amorphousfoam.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.12 (dd, 1H, 19-H, J=11Hz and 18 Hz), 5.54 (d, 1H, 14-H, J=8 Hz), 5.07 (m, 2H, 20-H), 5.00 (t,1H, 2′-OH, J=5 Hz), 4.78 (m, 1H, 5′-H), 4.50 (d, 1H, 11-OH, J=6 Hz),3.55-3.25 (m, 4H, 2′-H, 11-H, 22-H), 2.91 (m, 1H, 1′-H), 2.38 (bs, 4-H),1.36 (s, 3H, 15-CH₃), 1.05 (s, 3H, 18-CH₃), 0.80 (d, 3H, 17-CH₃, J=7Hz), 0.63 (d, 3H, 16-CH₃, J=7 Hz).

Step E.14-O-{[(1R,2R,5R)-5-Azido-2-hydroxy-5-methanesulfonyloxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5S)diastereomer

A solution of14-O-{[(1R,2R,5S)-2-hydroxy-5-methanesulfonyloxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (2.55 g, 4.35 mmol) and sodium azide (0.85 g, 13 mmol) in30 ml of dimethylformamide was heated for 6 hours at 80° C. The reactionmixture was diluted with water and brine and extracted three times withethyl acetate. The combined organic layers were washed with water andbrine, dried over sodium sulfate and filtered. The solvent was removedunder reduced pressure and crude14-O-{[(1R,2R,5R)-5-azido-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5S)diastereomer (quantitative yield, cyclohexane/ethyl acetate=1/1,R_(f)=0.35) was obtained as amorphous foam which was directly used forthe next step.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.15, 6.13 (2dd, 1H,19-H, J=11 Hz and 18 Hz), 5.56, 5.54 (2d, 1H, 14-H, J=8 Hz), 5.05 (m,2H, 20-H), 4.90 (d, 1H, 2′-OH, J=5 Hz), 4.50, 4.49 (2d, 1H, 11-OH, J=6Hz), 3.50-3.25 (m, 5H, 2′-H, 5′-H, 11-H, 22-H), 2.64 (m, 1H, 1′-H), 2.40(bs, 1H, 4-H), 1.36 (s, 3H, 15-CH₃), 1.06 (s, 3H, 18-CH₃), 0.81 (d, 3H,17- CH₃, J=7 Hz), 0.63 (d, 3H, 16-CH₃, J=7 Hz).

Step F.14-O-{[(1R,2R,5R)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5S)diastereomer

Triphenylphosphine (1.18 g, 4.50 mmol) was added to a solution of14-O-{[(1R,2R,5R)-5-azido-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5S)diastereomer (2.4 g, 4.50 mmol) in 30 ml of tetrahydrofuran and stirredovernight at room temperature. Subsequently water (approx. 3 ml) wasadded and the reaction mixture was heated for 1 hour at reflux. Afterevaporation of the solvent the residue was diluted with water and brineand extracted three times with ethyl acetate. The combined organiclayers were dried over sodium sulfate, filtered and subjected tochromatography (silica, ethyl acetate/methanol/35% ammoniasolution=100/100/1) to yield14-O-{[(1R,2R,5R)-5-amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5S)diastereomer (R_(f)=0.3, 1.74 g, 79% yield) as colorless amorphous foam.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 7.25, 6.65 (2bs, 1H, NH),6.14 (dd, 1H, 19-H, J=11 Hz and 18 Hz), 5.54 (d, 1H, 14-H, J=8 Hz), 5.04(m, 2H, 20-H), 4.50 (bs, 1H, 11-OH), 3.55-3.10 (m, 5H, 2′-H, 5′-H, 11-H,22-H), 2.58 (m, 1H, 1′-H), 2.40 (bs, 1H, 4-H), 1.36 (s, 3H, 15-CH₃),1.05 (s, 3H, 18-CH₃), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.62 (d, 3H, 16-CH₃, J=7 Hz). MS-ESI (m/z): 508 (MH⁺), 530 (MNa⁺), 1037 (2MNa⁺).

Example4A—14-O-{[(1R,2R,5R)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinand14-O-{[(1S,2S,5S)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin

The mixture of14-O-{[(1R,2R,5R)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5S)diastereomer (4 g, 7.87 mmol) from Example 4 Step F was separated on achiral column (250×20 mm CHIRALPAK IC,n-heptane/tetrahydrofuran/diethylamine=70/30/0.1) to yield14-O-{[(1S*,2S*,5S*)-5-amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin(late eluting compound, 1.1 g, 28% yield, uncorrected) as colorlessamorphous foam.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.13 (dd, 1H, 19-H, J=11Hz and 18 Hz), 5.54 (d, 1H, 14-H, J=8 Hz), 5.06 (m, 2H, 20-H), 5.76 (d,1H, 2′-OH, J=7 Hz), 4.50 (d, 1H, 11-OH, J=7 Hz), 3.55-3.15 (m, 5H, 2′-H,11-H, 5′-H, 22-H), 2.48 (m, 1H, 1′-H), 2.40 (bs, 1H, 4-H), 1.36 (s, 3H,15-CH₃), 1.05 (s, 3H, 18-CH₃), 0.81 (d, 3H, 17-CM₃, J=7 Hz), 0.62 (d,3H, 16-CH₃, J=7 Hz). MS-ESI (m/z): 508 (MH⁺), 1037 (2MNa⁺), 506 (M-H)⁻,542 (MCl⁻).

Example5—14-O-{[(1R,2R,3R)-3-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,3S) diastereomer hydrochloride Step A.14-O-{[(1R,2R,3R)-3-tert-Butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3S)diastereomer

(cis)-2,3-Epoxycyclohexyl-carbamic acid tert-butyl ester (O'Brien, P.;Childs, A., C.; Ensor, G. Organic Letters 2003, 5(26), 4955-4957.) (1 g,4.69 mmol) was treated with pleuromutilin thiol according to the methodof Example 1 Step A 1. After work up and chromatography of the reactionmixture (silica, cyclohexane/ethyl acetate=1/1)14-O-{[(1R,2R,3R)-3-tert-butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3S)diastereomer (R_(f)=0.5, 1.32 g, 46%) was obtained as colorlessamorphous foam.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.12 (m, 2H, NH, 19-Hz),5.54 (d, 1H, 14-H, J=8 Hz), 5.05 (m, 2H, 20-H), 4.96 (d, 1H, 2′-OH, J=4Hz), 4.50, 4.99 (2d, 1H, 11-OH, J=6 Hz)), 3.65 (m, 1H, 2′-H), 3.57 (m,1H, 3′-H), 3.42 (t, 1H, 11-H, J=6 Hz), AB-system (ν_(A)=3.30, 3.29,ν_(B)=3.23, 3.22, 22-H, J=15 Hz), 3.06 (m, 1H, 1′-H), 2.40 (bs, 1H,4-H), 1.36 (s, 12H, tert-butyl, 15-CH₃), 1.05 (s, 3H, 18-CH₃), 0.82 (d,3H, 17-CH₃, J=7 Hz), 0.63 (d, 3H, 16-CH₃, J=6 Hz).

Step B.14-O-{[(1R,2R,3R)-3-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3S)diastereomer

14-O-{[(1R,2R,3R)-3-tert-Butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3S)diastereomer (400 mg, 0.658 mmol) was treated according to the method ofExample 1 Step B. After work up and chromatography of the reactionmixture (silica, ethyl acetate/methanol=1/5)14-O-{[(1R,2R,3R)-3-amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3S)diastereomer (R_(f)=0.1, 249 mg, 75%) was obtained as colorlessamorphous foam.

MS-ESI (m/z): 508 (MH⁺), 530 (MNa⁺), 1015 (2MH⁺), 1037 (2MNa⁺).

Step C.14-O-{[(1R,2R,3R)-3-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,4S) diastereomer hydrochloride

14-O-{[(1R,2R,3R)-3-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3S)diastereomer (249 mg, 0.49 mmol) was treated according to the method ofExample 1 Step C to obtain14-O-{[(1R,2R,3R)-3-amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,3S) diastereomer hydrochloride (247 mg, 93% yield)as colorless amorphous solid.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 7.8 (bs, 3H, NH₃ ⁺), 6.13(d, 2H, 19-Hz, J=11 Hz and 18 Hz), 5.80 (d, 1H, 2′-OH, J=4 Hz), 5.55 (d,1H, 14-H, J=8 Hz), 5.05 (m, 2H, 20-H), 4.55, 4.54 (2d, 1H, 11-OH, J=6Hz)), 3.87 (m, 1H, 2′-H), 3.42 (t, 1H, 11-H, J=6 Hz), AB-system(ν_(A)=3.35, ν_(B)=3.24, 22-H, J=15 Hz), 3.20, 3.13 (2m, 1H, 3′-H,1′-H), 2.40 (bs, 1H, 4-H), 1.36 (s, 3H, 15-CH₃), 1.05 (s, 3H, 18-CH₃),0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.63 (d, 3H, 16-CH₃, J=7 Hz). MS-ESI(m/z): 508 (MH⁺), 1015 (2MH⁺), 542 (MCl⁻).

Example6—14-O-{[(1R,2R,4R)-4-Diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S)diastereomer and14-O-{[(1R,2R,4R)-4-Ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S)diastereomer

To a solution of14-O-{[(1R,2R,4R)-4-amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S)diastereomer (900 mg, 1.77 mmol) from Example 1 Step B in 10 mldichloromethane was added acetaldehyde (2.77 ml, 1M in dichloromethane)and acetic acid (77 μl, 1.77 mmol) and stirred for 30 minutes at roomtemperature. The resulting reaction mixture was treated with sodiumtriacetoxyborohydride (750 mg, 3.54 mmol) and stirred overnight at roomtemperature, diluted with dichloromethane and subsequently extractedwith NaHCO₃ solution and brine. The organic layer was dried over sodiumsulfate and filtered. The filtrate was subjected to chromatography(silica, ethyl acetate/methanol/35% ammonia solution=50/50/1) to yield14-O-{[(1R,2R,4R)-4-diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S)diastereomer (a) (92 mg, 9% yield) and14-O-{[(1R,2R,4R)-4-ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S)diastereomer (b) (163 mg, 17% yield) as colorless amorphous foams.

(a): ¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.14 (dd, 1H, 19-H,J=11 Hz and 18 Hz), 5.55 (d, 1H, 14-H, J=8 Hz), 5.05 (m, 2H, 20-H), 4.83(d, 1H, 2′-OH, J=4 Hz), 4.47 (d, 1H, 11-OH, J=6 Hz), 3.42 (m, 1H, 11-H),AB-system (ν_(A)=3.50, 3.42, ν_(B)=3.30, 3.27, 22-H, J=15 Hz), 3.25 (m,1H, 2′-H), 2.50 (m, 2H, 1′-H, 4′-H), 2.40 (m, 5H, NCH₂, 4-H), 1.36 (s,3H, 15-CH₃), 1.05 (s, 3H, 18-CH₃), 0.92 (t, 6H, NCH₂CH₃, J=7 Hz), 0.81(d, 3H, 17- CH₃, J=7 Hz), 0.63 (d, 3H, 16-CH₃, J=7 Hz). MS-ESI (m/z):564 (MH⁺), 586 (MNa⁺), 562 (M-H)⁻.

(b): ¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.13 (dd, 1H, 19-H,J=11 Hz and 18 Hz), 5.54 (d, 1H, 14-H, J=8 Hz), 5.05 (m, 2H, 20-H), 4.48(d, 1H, 11-OH, J=6 Hz), 3.42 (m, 1H, 11-H), AB-system (ν_(A)=3.48,ν_(B)=3.25, 22-H, J=15 Hz), 2.55 (m, 2H, 1′-H, 4′-H), 2.40 (bs, 1H,4-H), 1.35 (s, 3H, 15-CH₃), 1.05 (s, 3H, 18-CH₃), 0.95 (t, 3H, NCH₂CH₃,J=7 Hz), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.63 (d, 3H, 16-CH₃, J=6 Hz).MS-ESI (m/z): 536 (MH⁺), 558 (MNa⁺), 534 (M-H)⁻.

Example7—14-O-{[(1R,2R,5S)-5-Ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,5R) diastereomer hydrochloride Step A.N-Ethyl-N-(cyclohex-3-enyl)-carbamic acid tert-butyl ester

To a solution of cyclohex-3-enyl-carbamic acid tert-butyl ester(Kampferer, P.; Vasella, A. Helvetica Chimica Acta 2004, 87, 2764-2789)(4.34 g, 22 mmol) in 20 ml of DMSO was added sodium hydride (880 mg, 60%dispersion, 22 mmol) and after one hour of stirring ethyl iodide (1.78ml, 22 mmol). After further stirring for 2 hours at room temperature thereaction mixture was concentrated under reduced pressure. The residuewas diluted with water and brine and extracted three times with ethylacetate. The organic layers were dried over sodium sulfate and filtered.The filtrate was concentrated under reduced pressure and afterchromatography (silica, cyclohexane/ethyl acetate=12/1) the titlecompound (R_(f)=0.30, 2.88 g, 58% yield) was obtained as colorlesssolid.

¹H NMR (400 MHz, CDCl₃, δ, ppm): 5.61 (m, 2H, double bond), 4.08 (bs,1H, NCH), 3.15 (m, 2H, NCH₂), 2.15, 1.75 (2m, 6H), 1.47 (s, 9H,tert-butyl), 1.13 (t, 3H, NCH₂CH₃, J=7 Hz).

Step B. N-ethyl-N-(cis-3,4-epoxycyclohexyl)-carbamic acid tert-butylester

N-Ethyl-N-(cyclohex-3-enyl)-carbamic acid tert-butyl ester (2.87 g, 12.7mmol) was dissolved in 75 ml of dichloromethane and treated with3-chloroperbenzoic acid (4.50 g, 70%, 19 mmol). After stirring at roomtemperature for 20 hours the reaction mixture was concentrated underreduced pressure. The residue was diluted with ethyl acetate andsubsequently extracted with 10% NaHSO₃ solution, saturated NaHCO₃solution and brine. The organic layer was dried over sodium sulfate andfiltered. The filtrate was concentrated under reduced pressure and afterchromatography (silica, cyclohexane/dioxane=5/1) the title compound(R_(f)0.2, 1.50 g, 49% yield) was obtained.

¹H NMR (400 MHz, CDCl₃, δ, ppm): 4.0 (bs, 1H, NCH), 3.14 (m, 2H, NCH₂),3.06 (bs, 2H, epoxide), 2.13, 2.08, 1.88, 1.60, 1.36 (4m, 6H), 1.47 (s,9H, tert-butyl), 0.08 (t, 3H, NCH₂CH₃).

Step C.14-O-{[(1R,2R,5S)-5-(tert-Butoxycarbonyl-ethyl-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer

N-Ethyl-N-(cis-3,4-epoxycyclohexyl)-carbamic acid tert-butyl ester (1.5g, 6.2 mmol) was treated with pleuromutilin thiol according to themethod of Example 1 Step A1. After work up and chromatography of thereaction mixture (silica, cyclohexane/dioxane=3/1)14-O-{[(1R,2R,5S)-5-(tert-butoxycarbonyl-ethyl-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (R_(f)=0.4, 2.57 g, 65% yield) was obtained as colorlessamorphous foam.

MS-ESI (m/z): 536 (MH⁺), 558 (MNa⁺), 534 (M-H)⁻.

Step D.14-O-{[(1R,2R,5S)-5-Ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+1S,2S,5R)diastereomer

14-O-{[(1R,2R,5S)-5-(tert-Butoxycarbonyl-ethyl-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (2.57 g, 4.04 mmol) was treated according to the method ofExample 1 Step B. After work up and chromatography of the reactionmixture (silica, ethyl acetate/methanol/35% ammonia solution=100/100/1)14-O-{[(1R,2R,5S)-5-ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (R_(f)=0.3, 1.08 g, 50%) was obtained as colorlessamorphous foam.

¹H NMR (500 MHz, CDCl₃, δ, ppm, inter alia): 6.48 (dd, 1H, 19-H, J=10 Hzand 18 Hz), 5.77 (m, 1H, 14-H), 5.36 (m, 1H, 20-H), 5.22 (d, 1H, 20-H,J=17 Hz), 3.45 (d, 1H, 2′-H), 3.37 (d, 1H, 11-H, J=6 Hz), 3.25 (m, 1H,22-H), 2.97 (m, 1H, 1′-H), 2.91 (m, 1H, 5′-H), 2.63 (q, 2H, NCH₂, J=7Hz), 2.10 (bs, 1H, 4-H), 1.46 (s, 3H, 15-CH₃), 1.18 (s, 3H, 18-CH₃),1.12 (t, 3H, NCH₂CH₃, J=7 Hz), 0.98 (d, 3H, 17-CH₃, J=7 Hz), 0.73 (d,3H, 16-CH₃, J=7 Hz).

Step E.14-O-{[(1R,2R,5S)-5-Ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,5R) diastereomer hydrochloride

14-O-{[(1R,2R,5S)-5-Ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (86 mg, 0.16 mmol) was treated according to the method ofExample 1 Step C to obtain14-O-{[(1R,2R,5S)-5-ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,5R) diastereomer hydrochloride (83 mg, 90% yield)as colorless amorphous solid.

¹H NMR (400 MHz, CDCl₃, δ, ppm, inter alia): 9.3 (bs, 2H, NH₂ ⁺), 6.45(m, 1H, 19-H), 5.73 (d, 1H, 14-H, J=10 Hz), 5.35 (m, 1H, 20-H), 5.22 (d,1H, 22-H, J=18H), 3.85 (m, 1H, 2′-H), 3.33 (m, 3H, 11-H, 22-H), 3.07 (m,2H, NCH₂), 2.10 (bs, 1H, 4-H), 1.50 (t, 3H, NCH₂CH₃, J=7 Hz), 1.45 (s,3H, 15-CH₃), 1.18 (s, 3H, 18-CH₃), 0.90 (d, 3H, 17-CH₃, J=7 Hz), 0.73(d, 3H, 16-CH₃, J=7 Hz). MS-ESI (m/z): 536 (MH⁺), 570 (MCl⁻).

Example8—14-O-{[(1R,2R,5S)-5-Diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,5R) diastereomer hydrochloride Step A.14-O-{[(1R,2R,5S)-5-Diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer

14-O-{[(1R,2R,5S)-5-Ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (250 mg, 0.47 mmol) from Example 7 Step D was treated withacetaldehyde (53 μl, 0.93 mmol) according to the method of Example 6.After work up and chromatography of the reaction mixture (silica, ethylacetate/methanol=1/1)14-O-{[(1R,2R,5S)-5-diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (R_(f)0.2, 230 mg, 87%) was obtained as colorless amorphousfoam.

¹H NMR (400 MHz, CDCl₃, δ, ppm, inter alia): 6.48 (dd, 1H, 19-H, J=11 Hzand 17 Hz), 5.77 (d, 1H, 14-H, J=8 Hz), 5.36 (m, 1H, 20-H), 5.22 (d, 1H,20-H, J=17 Hz), 3.57, 3.36, 3.21, 3.03, 2.72 (5m, 6H, 1′-H, 5′-H, 11-H,22-H), 2.59 (m, 4H, NCH₂), 2.11 (bs, 1H, 4-H), 1.46 (s, 3H, 15-CH₃),1.18 (s, 3H, 18-CH₃), 0.98 (t, 6H, NCH₂CH₃, J=7 Hz), 0.88 (d, 3H, 17-CH₃, J=7 Hz), 0.73 (d, 3H, 16-CH₃, J=7 Hz).

Step B.14-O-{[(1R,2R,5S)-5-Diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,5R) diastereomer hydrochloride

14-O-{[(1R,2R,5S)-5-Diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (230 mg, 0.41 mmol) was treated according to the method ofExample 1 Step C to obtain14-O-{[(1R,2R,5S)-5-diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,5R) diastereomer hydrochloride (223 mg, 91% yield)as colorless amorphous solid.

¹H NMR (400 MHz, CDCl₃, δ, ppm, inter alia): 11.5 (bs, 3H, NH⁺), 6.46(dd, 1H, 19-H, J=11 Hz and 17 Hz), 5.74 (d, 1H, 14-H, J=8 Hz), 5.34 (m,1H, 20-H), 5.22 (d, 1H, 22-H, J=17H), 3.98 (m, 1H, 2′-H), 3.60-2.90 (m,9H, 1′-H, 5′-H, 11-H, 22-H, NCH₂), 2.10 (bs, 1H, 4-H), 1.48 (m, 9H,NCH₂CH₃, 15-CH₃), 1.18 (s, 3H, 18-CH₃), 0.89 (d, 3H, 17-CH₃, J=7 Hz),0.73 (d, 3H, 16-CH₃, J=7 Hz). MS-ESI (m/z): 564 (MH⁺), 586 (MNa⁺), 1149(2MNa⁺), 598 (MCl⁻).

Example9—14-O-{[(1R,2R,4S)-4-Diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R)diastereomer

14-O-{[(1R,2R,4S)-4-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R)diastereomer (680 mg, 1.34 mmol) from Example 2 Step A was treatedaccording to the method of Example 6. After work up and chromatographyof the reaction mixture (silica, ethyl acetate/methanol=1/1)14-O-{[(1R,2R,4S)-4-diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R)diastereomer (R_(f)0.2, 129 mg, 17%) was obtained as colorless amorphousfoam.

¹H NMR (500 MHz, DMSO-d₆, δ, ppm, inter alia): 6.13 (dd, 1H, 19-H, J=11Hz and 18 Hz), 5.54 (d, 1H, 14-H, J=8 Hz), 5.05 (m, 2H, 20-H), 4.70 (d,1H, 2′-OH), 4.49 (d, 1H, 11-OH, J=6 Hz), 3.70 (m, 1H, 2′-H), 3.42 (t,1H, 11-H, J=6 Hz), AB-system (ν_(A)=3.36, ν_(B)=3.22, 22-H, J=15 Hz),2.72 (m, 2H, 1′-H, 4′-H), 2.47 (m, 2H, NCH₂), 2.40 (bs, 1H, 4-H), 1.35(s, 3H, 15-CH₃), 1.04 (s, 3H, 18-CH₃), 0.95 (t, 3H, NCH₂CH₃, J=7 Hz),0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.62 (d, 3H, 16-CH₃, J=7 Hz). MS-ESI(m/z): 536 (MH⁺), 558 (MNa⁺), 534 (M-H)⁺.

Example10—14-O-{[(1R,2R,5R)-5-Diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5S)diastereomer

14-O-{[(1R,2R,5R)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5S)diastereomer (420 mg, 0.827 mmol) from Example 4 Step F was treatedaccording to the method of Example 6. After work up and chromatographyof the reaction mixture (silica, ethyl acetate/methanol/35% ammoniasolution=50/50/1)14-O-{[(1R,2R,5R)-5-diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5S)diastereomer (R_(f)=0.2, 95 mg, 20%) was obtained as colorless amorphousfoam.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.13 (dd, 1H, 19-H, J=11Hz and 18 Hz), 5.55 (d, 1H, 14-H, J=8 Hz), 5.05 (m, 2H, 20-H), 4.79 (m,1H, 2′-OH), 3.55-3.15 (m, 2′-H, 5′-H, 11-H, 22-H), 2.58 (m, 1H, 1′-H),2.40 (m, 5H, NCH₂, 4-H), 1.37 (s, 3H, 15-CH₃), 1.06 (s, 3H, 18-CH₃),0.92 (t, 3H, NCH₂CH₃, J=7 Hz), 0.83 (d, 3H, 17-CH₃, J=7 Hz), 0.63 (d,3H, 16-CH₃, J=7 Hz). MS-ESI (m/z): 564 (MH⁺), 586 (MNa⁺), 562 (M-H)⁻,598 (MCl⁻).

Example11—14-O-{[(1R,2R,3R)-3-Ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3S)diastereomer Step A.[(1R,2R,3R)-2-Hydroxy-3-(2,4,6-trimethyl-benzylsulfanyl)-clohexyl]-carbamicacid tert-butyl ester+(1S,2S,3S) diastereomer

To a solution of (cis)-2,3-epoxycyclohexyl-carbamic acid tert-butylether (O'Brien, P.; Childs, A., C.; Ensor, G. Organic Letters 2003,5(26), 4955-4957.) (14.9 g, 68.9 mmol) and 2,4,6-trimethylbenzylmercaptan (11.5 g, 68.9 mmol) in 50 ml of methanol was added 10N NaOH (5ml, 50 mmol) and the resulting mixture was stirred for 16 hours at roomtemperature. The reaction mixture was diluted with water and brine andextracted with ethyl acetate three times. The organic layers were driedover sodium sulfate and filtered. The filtrate was subjected tochromatography (silica, cyclohexane/ethyl acetate=5/1) to yield[(1R,2R,3R)-2-hydroxy-3-(2,4,6-trimethyl-benzylsulfanyl)-cyclohexyl]-carbamicacid tert-butyl ester+(1S,2S,3S) diastereomer (R_(f)=0.25, 5.92 g, 23%yield) as colorless amorphous foam.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm): 6.78 (s, 2H, aromat.-H), 6.15 (bd,OH), 4.95 (bd, NH), 3.75 (d, 1H, SCH₂), 3.68 (m, 2H), 3.02 (m, 1H, SCH),2.30 (s, 9H, CH₃), 2.30, 1.90, 1.40 (3m, 6H), 1.35 (s, 9H, tert-butyl).

Step B.[(1R,2R,3R)-2-(tert-Butyl-dimethyl-silanyloxy)-3-(2,4,6-trimethyl-benzylsulfanyl)-cyclohexyl]-carbamicacid tert-butyl ester+(1S,2S,3S) diastereomer

A solution of[(1R,2R,3R)-2-hydroxy-3-(2,4,6-trimethyl-benzylsulfanyl)-cyclohexyl]-carbamicacid tert-butyl ester+(1S,2S,3S) diastereomer (2.46 g, 6.49 mmol) in 50ml of dimethylformamide was treated with tert-butyldimethylsilylchloride (978 mg, 6.49 mmol) and imidazole (552 mg, 8.11 mmol) andstirred at 80° C. for 5 days. The reaction mixture was concentratedunder reduced pressure. The residue was diluted with 0.1 N HCl andextracted three times with ethyl acetate. The combined organic layerswere washed with brine, dried over sodium sulfate and filtered. Afterchromatography (silica, cyclohexane/ethyl acetate=10/1)[(1R,2R,3R)-2-(tert-butyl-dimethyl-silanyloxy)-3-(2,4,6-trimethyl-benzylsulfanyl)-cyclo-hexyl]-carbamicacid tert-butyl ester+(1S,2S,3S) diastereomer (R_(f)=0.25, 3.0 g, 94%yield) was obtained.

¹H NMR (500 MHz, DMSO-d₆, δ, ppm): 6.80 (s, 2H, aromat-H), 6.20 (bd,NH), 3.90, 3.75, 3.63 (3m, 4H, NCH, OCH, SCH₂), 2.98 (m, 1H, SCH), 2.30(s, 9H, CH₃), 1.90, 1.50, 1.33 (3m, 6H), 1.35 (s, 9H, C-tert-butyl),0.85 (s, 9H, Si-tert-butyl), 0.0 (s, 6H, Si(CH₃)₂).

Step C.[(1R,2R,3R)-2-(tert-Butyl-dimethyl-silanyloxy)-3-(2,4,6-trimethyl-benzylsulfanyl)-cyclohexyl]-ethyl-carbamicacid tert-butyl ester+(1S,2S,3S) diastereomer

A solution of[(1R,2R,3R)-2-(tert-butyl-dimethyl-silanyloxy)-3-(2,4,6-trimethyl-benzylsulfanyl)-cyclohexyl]-carbamicacid tert-butyl ester+(1S,2S,3S) diastereomer (3.0 g, 6.08 mmol) wastreated with ethyl iodide according to the method of Example 7 Step A.After work up and chromatography of the reaction mixture (silica,cyclohexane/ethyl acetate=3/1)[(1R,2R,3R)-2-(tert-butyl-dimethyl-silanyloxy)-3-(2,4,6-trimethyl-benzylsulfanyl)-cyclohexyl]-ethyl-carbamicacid tert-butyl ester+(1S,2S,3S) diastereomer (1.20 g, 38%) wasobtained.

MS-ESI (m/z): 544 (MNa⁺), 1065 (2MNa⁺).

Step D.[(1R,2R,3R)-2-(tert-Butyl-dimethyl-silanyloxy)-3-mercapto-cyclohexyl]-ethyl-carbamicacid tert-butyl ester+(1S,2S,3S) diastereomer

A solution of[(1R,2R,3R)-2-(tert-butyl-dimethyl-silanyloxy)-3-(2,4,6-trimethyl-benzylsulfanyl)-cyclohexyl]-ethyl-carbamicacid tert-butyl ester+(1S,2S,3S) diastereomer (1.20 g, 2.30 mmol) in 10ml of tetrahydrofuran and 20 ml of liquid ammonia was treated at −78° C.under an argon atmosphere with sodium (106 mg, 4.60 mmol) and stirred at−78° C. for one hour. Then solid ammonium chloride was added and thereaction mixture was warmed to room temperature, diluted withtetrahydrofuran and flushed with nitrogen. The residual mixture wasfiltered and concentrated under reduced pressure to yield crude[(1R,2R,3R)-2-(tert-butyl-dimethyl-silanyloxy)-3-mercapto-cyclohexyl]-ethyl-carbamicacid tert-butyl ester+(1S,2S,3S) diastereomer (quantitative yield) whichwas directly used for the next step.

MS-ESI (m/z): 412 (MNa⁺), 801 (2MNa⁺).

Step E.14-O-{[(1R,2R,3R)-3-(tert-Butoxycarbonyl-ethyl-amino)-2-(tert-butyl-dimethyl-silanyloxy)-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3S)diastereomer

Crude[(1R,2R,3R)-2-(tert-butyl-dimethyl-silanyloxy)-3-mercapto-cyclohexyl]-ethyl-carbamicacid tert-butyl ester+(1S,2S,3S) diastereomer (895 mg, 2.30 mmol) wasdissolved in 30 ml of tetrahydrofuran and treated subsequently withpleuromutilin tosylate (979 mg, 1.84 mmol) and potassium tert-butoxide(206 mg, 1.84 mmol) and the resulting mixture was stirred for 16 hoursat room temperature. After evaporation of the solvent the residue wasdiluted with 1N HCl and extracted three times with ethyl acetate. Thecombined organic layers were washed with NaHCO₃ solution and brine,dried over sodium sulfate and filtered. After chromatography (silica,cyclohexane/ethyl acetate=10/1)14-O-{[(1R,2R,3R)-3-(tert-butoxycarbonyl-ethyl-amino)-2-(tert-butyl-dimethyl-silanyloxy)-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3S)diastereomer (R_(f)=0.5, 468 mg, 27% yield) was obtained.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.14 (dd, 1H, 19-H, J=11Hz and 18 Hz), 5.55 (d, 1H, 14-H, J=8 Hz), 5.05 (m, 2H, 20-H), 4.49 (m,1H, 11-OH), 3.94 (m, 1H, 2′-H), 3.43 (t, 1H, 11-H, J=6 Hz), 3.28, 3.04(2m, 5H, 1′-H, 22-H, NCH₂), 2.40 (bs, 1H, 4-H), 1.40 (s, 9H,tert-butyl), 1.36, 1.35 (2s, 3H, 15-CH₃), 1.05 (s, 3H, 18-CH₃), 0.87 (s,9H, Si-tert-butyl), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.64, 0.62 (2d, 3H,16-CH₃, J=7 Hz) 0.05, -0.05 (2s, 6H, Si(CH₃)₂).

Step F.14-O-{[(1R,2R,3R)-3-Ethylamino-2-(tert-butyl-dimethyl-silanyloxy)-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3S)diastereomer and14-O-{[(1R,2R,3R)-3-Ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3S)diastereomer

14-O-{[(1R,2R,3R)-3-(tert-Butoxycarbonyl-ethyl-amino)-2-(tert-butyl-dimethyl-silanyloxy)-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3S)diastereomer (468 mg, 0.624 mmol) was treated with trifluoroacetic acidovernight according to the method of Example 1 Step B. After work up andchromatography of the reaction mixture (silica, ethylacetate/methanol=1/2)14-O-{[(1R,2R,3R)-3-ethylamino-2-(tert-butyl-dimethyl-silanyloxy)-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3S)diastereomer (a) (R_(f)=0.6, 144 mg, 36% yield) and14-O-{[(1R,2R,3R)-3-ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3S)diastereomer (b) (R_(f)=0.25, 177 mg, 53% yield) were obtained ascolorless solids.

(a): MS-ESI (m/z): 672 (MNa⁺), 1321 (2MNa⁺).

(b): ¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.14 (dd, 1H, 19-H,J=11 Hz and 18 Hz), 5.54 (d, 1H, 14-H, J=8 Hz), 5.05 (m, 2H, 20-H), 4.76(m, 1H, 2′-OH), 4.49, 4.48 (2d, 1H, 11-OH, J=6 Hz), 3.55 (m, 1H, 2′-H),3.42 (t, 1H, 11-H, J=6 Hz), AB-system (ν_(A)=3.37, ν_(B)3.18, 22-H, J=15Hz), 3.05 (m, 1H, 3′-H), 2.66 (m, 1H, 1′-H), 2.50 (m, 2H, NCH₂), 2.40(bs, 1H, 4-H), 1.36 (s, 3H, 15-CH₃), 1.05 (s, 3H, 18-CH₃), 0.98 (t, 3H,NCH₂CH₃, J=7 Hz), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.62 (d, 3H, 16-CH₃, J=7Hz). MS-ESI (m/z): 536 (MH⁺), 558 (MNa⁺), 1071 (2MH⁺), 1093 (2MNa⁺), 534(M-H)⁻.

Example12—14-O-{[(1R,2R,3R)-3-Diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3S)diastereomer Step A.14-O-{[(1R,2R,3R)-3-Diethylamino)-2-(tert-butyl-dimethyl-silanyloxy)-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3S)diastereomer

14-O-{[(1R,2R,3R)-3-Ethylamino-2-(tert-butyl-dimethyl-silanyloxy)-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3S)diastereomer (144 mg, 0.222 mmol) from Example 11 Step F was treatedwith acetaldehyde (25 μl, 0.444 mmol) according to the method of Example6. After work up and chromatography of the reaction mixture (silica,ethyl acetate/methanol=2/1)14-O-{[(1R,2R,3R)-3-diethylamino)-2-(tert-butyl-dimethyl-silanyloxy)-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3S)diastereomer (R_(f)=0.5, 110 mg, 73%) was obtained as colorlessamorphous foam.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.14 (dd, 1H, 19-H, J=11Hz and 18 Hz), 5.53 (d, 1H, 14-H, J=7 Hz), 5.05 (m, 2H, 20-H), 4.98 (m,1H, 11-OH), 3.97 (m, 1H, 2′-H), 3.42 (t, 1H, 11-H, J=6 Hz), 3.24 (m, 2H,H-22), 3.00 (m, 1H, 1′-H), 2.70 (m, 1H, 3′-H), 2.55 (m, 4H, NCH₂), 2.40(bs, 1H, 4-H), 1.36 (s, 3H, 15-CH₃), 1.05 (s, 3H, 18-CH₃), 0.87 (m, 15H,NCH₂CH₃, Si-tert-butyl), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.62, 0.60 (2d,3H, 16-CH₃, J=7 Hz), 0.07 (s, 6H, Si(CH₃)₂).

Step B.14-O-{[(1R,2R,3R)-3-Diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3S)diastereomer

To a solution of14-O-{[(1R,2R,3R)-3-diethylamino-2-(tert-butyl-dimethyl-silanyloxy)-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3S)diastereomer (101 mg, 0.149 mmol) in 5 ml of tetrahydrofuran was addedtetrabutylammonium fluoride (0.44 ml, 1M in tetrahydrofuran, 0.447mmol). After stirring at room temperature for 2 days the reactionmixture was concentrated under reduced pressure, diluted with NaHCO₃solution and extracted two times with ethyl acetate. The combinedorganic layers were dried over sodium sulfate and filtered. The filtratewas subjected to chromatography (silica, ethyl acetate/methanol=1/2) toobtain14-O-{[(1R,2R,3R)-3-diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3S)diastereomer (R_(f)=0.2, 8 mg, 10% yield) as colorless amorphous foam.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.15, 6.14 (2dd, 1H,19-H, J=11 Hz and 18 Hz), 5.54 (d, 1H, 14-H, J=8 Hz), 5.05 (m, 2H,20-H), 4.99, 4.42 (2m, 2H, 11-OH, 2′-OH), 3.87 (m, 1H, 2′-H), 3.42 (t,1H, 11-H, J=6 Hz), 3.25 (m, 2H, H-22), 3.05 (m, 1H, 1′-H), 2.60 (m, 3H,3′-H, NCH₂), 2.40 (bs, 1H, 4-H), 1.35 (s, 3H, 15-CH₃), 1.05 (s, 3H,18-CH₃), 0.90 (m, 6H, NCH2CH₃), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.62, 0.61(2d, 3H, 16-CH₃, J=7 Hz).

MS-ESI (m/z): 564 (MH⁺), 586 (MNa⁺), 1149 (2MNa⁺), 562 (M-H)⁻.

Example13—14-O-{[(1R,2R,4S)-4-(Formyl-hydroxy-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R)diastereomer Step A1.14-O-{[(7R,8R)-7-Hydroxy-1,4-dioxa-spiro[4.5]dec-8-ylsulfanyl]-acetyl}-mutilin+(7S,8S)diastereomer

7,8-Epoxy-1,4-dioxa-spiro[4.5]decane (Zhang, L.; Koreeda, M. OrganicLetters 2002, 4(21), 3755-3758.) (6.25 g, 40 mmol) and pleuromutilinthiol (8 g, 20 mmol) were treated according to the method of Example 1Step A2. After work up and chromatography of the reaction mixture(silica, cyclohexane/ethyl acetate=1/1)14-O-{[(7R,8R)-7-hydroxy-1,4-dioxa-spiro[4.5]dec-8-ylsulfanyl]-acetyl}-mutilin+(7S,8S)diastereomer (R_(f)0.3, 8.40 g, 76% yield) was obtained as colorlessamorphous foam.

¹H NMR (500 MHz, DMSO-d₆, δ, ppm, inter alia): 6.14 (dd, 1H, 19-H, J=11Hz and 18 Hz), 5.55 (d, 1H, 14-H, J=8 Hz), 5.05 (m, 2H, 20-H), 4.95 (d,1H, 2′-OH, J=6 Hz), 4.50 (d, 1H, 11-OH, J=6 Hz), 3.82 (m, 4H, OCH₂CH₂O),3.55-3.25 (m, 4H, 2′-H, 11-H, H-22), 2.58(m, 1H, 1′-H), 2.40 (bs, 1H,4-H), 1.35, 1.34 (2s, 3H, 15-CH₃), 1.05 (s, 3H, 18-CH₃), 0.81 (d, 3H,17-CH₃, J=7 Hz), 0.62 (d, 3H, 16-CH₃, J=7 Hz). MS-ESI (m/z): 573 (MNa⁺),549 (M-H)⁻.

or Step A2.14-O-{[(7R,8R)-7-Hydroxy-1,4-dioxa-spiro[4.5]dec-8-ylsulfanyl]-acetyl}-mutilin+(7S,8S)diastereomer and14-O-{[(7R,8R)-8-Hydroxy-1,4-dioxa-spiro[4.5]dec-7-ylsulfanyl]-acetyl}-mutilin+(7S,8S)diastereomer

7,8-Epoxy-1,4-dioxa-spiro[4.5]decane (6.24 g, 39.95 mmol) andpleuromutilin thiol (16.4 g, 41.7 mmol) were treated according to themethod of Example 1 Step A3. After work up and chromatography of thereaction (silica, ethyl acetate/toluene=1/1) a mixture of14-O-{[(7R,8R)-7-hydroxy-1,4-dioxa-spiro[4.5]dec-8-ylsulfanyl]-acetyl}-mutilin+(7S,8S)diastereomer as well as14-O-{[(7R,8R)-8-hydroxy-1,4-dioxa-spiro[4.5]dec-7-ylsulfanyl]-acetyl}-mutilin+(7S,8S)diastereomer (R_(f)=0.24, 4.40 g, 20% yield) was obtained as colorlesssolid.

¹H NMR (500 MHz, DMSO-d₆, δ, ppm, inter alia): 6.14, 6.12 (2dd, 1H,19-H, J=11 Hz and 18 Hz), 5.55, 5.53 (2d, 1H, 14-H, J=8Hz), 5.05 (m, 2H,20-H), 4.95, 4.87 (d+dd, 1H, 2′-OH, J=6 Hz), 4.50, 4.49 (2d, 1H, 11-OH,J=6 Hz), 3.83 (m, 4H, OCH₂CH₂O), 3.55-3.25 (m, 4H, 2′-H, 11-H, H-22),2.77, 2.57 (2m, 1H, 1′-H), 2.40 (bs, 1H, 4-H), 1.36, 1.36 (2s, 3H,15-CH₃), 1.05 (s, 3H, 18-CH₃), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.62 (d,3H, 16-CH₃, J=7 Hz). MS-ESI (m/z): 573 (MNa⁺), 549 (M-H)⁻.

Step B.14-O-{[(7R,8R)-7-(tert-Butyl-diphenyl-silanyloxy)-1,4-dioxa-spiro[4.5]dec-8-ylsulfanyl]-acetyl}-mutilin+(7S,8S)diastereomer

A solution of14-O-{[(7R,8R)-7-hydroxy-1,4-dioxa-spiro[4.5]dec-8-ylsulfanyl]-acetyl}-mutilin+(7S,8S)diastereomer (8.4 g, 15.3 mmol) from Step A1 in 50 ml ofdimethylformamide was treated with tert-butyldiphenylsilyl chloride(5.16 ml, 19.8 mmol) and imidazole (1.66 g, 24.4 mmol) and stirredovernight at 80° C. The reaction mixture was concentrated under reducedpressure. The residue was diluted with water and brine and extractedthree times with ethyl acetate. The combined organic layers were washedwith water and brine, dried over sodium sulfate and filtered. Afterchromatography (silica, cyclohexane/ethyl acetate=1/1)14-O-{[(7R,8R)-7-(tert-butyl-diphenyl-silanyloxy)-1,4-dioxa-spiro[4.5]dec-8-ylsulfanyl]-acetyl}-mutilin+(7S,8S)diastereomer (R_(f)=0.7, 8.03 g, 67% yield) was obtained.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 7.7-7.35 (m, 10H,aromat.-H), 6.15, 6.13 (2dd, 1H, 19-H, J=11 Hz and 18 Hz), 5.57, 5.53(2d, 1H, 14-H, J=7 Hz), 5.05 (m, 2H, 20-H), 4.50 (m, 1H, 11-OH), 3.30(m, 1H, 2′-H), 3.70-2.80 (m, 8H, OCH₂CH₂O, 1′-H, 11-H, 22-H), 2.40 (bs,1H, 4-H), 1.39, 1.36 (2s, 3H, 15-CH₃), 1.05 (s, 3H, 18-CH₃), 1.00 (s,9H, Si-tert-butyl), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.62, 0.60 (2d, 3H,16-CH₃, J=7 Hz). MS-ESI(m/z): 811 (MNa⁺).

Step C.14-O-{[(1R,2R)-2-(tert-Butyl-diphenyl-silanyloxy)-4-oxo-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer

14-O-{[(7R,8R)-7-(tert-Butyl-diphenyl-silanyloxy)-1,4-dioxa-spiro[4.5]dec-8-ylsulfanyl]-acetyl}-mutilin+(7S,8S)diastereomer (8.03 g, 10.2 mmol) was dissolved in 100 ml ofdichloromethane and treated with montmorillonite K10 (10 g) for 3 daysat room temperature. After filtration over celite the reaction mixturewas concentrated under reduced pressure and subjected to chromatography(silica, cyclohexane/ethyl acetate=2/1) to yield14-O-{[(1R,2R)-2-(tert-butyl-diphenyl-silanyloxy)-4-oxo-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer (R_(f)=0.38, 5.24 g, 69% yield).

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 7.65-7.40 (m, 10H,aromat.-H), 6.15, 6.13 (2dd, 1H, 19-H, J11 Hz and 18 Hz), 5.53 (d, 1H,14-H, J=7 Hz), 5.00 (m, 2H, 20-H), 4.47 (m, 1H, 11-OH), 4.24 (m, 1H,2′-H), 3.41 (t, 1H, 11-H, J=6 Hz), 3.20-3.00 (m, 2H, 22-H), 2.60 (m, 1H,1′-H), 2.40 (bs, 1H, 4-H), 1.35, 1.33 (2s, 3H, 15-CH₃), 1.06 (s, 3H,18-CH₃), 0.97 (s, 9H, Si-tert-butyl), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.58(d, 3H, 16-CH₃, J=7 Hz).

Step D.14-O-{[(1R,2R)-2-(tert-Butyl-diphenyl-silanyloxy)-4-hydroxyimino-cyclohexylsulfany]-acetyl}-mutilin+(1S,2S)diastereomer

To a solution of14-O-{[(1R,2R)-2-(tert-butyl-diphenyl-silanyloxy)-4-oxo-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer (2.50 g, 3.36 mmol) in 10 ml of dimethylformamide was addedhydroxylamine hydrochloride (233 mg, 3.36 mmol) and triethylamine (0.47ml, 3.36 mmol) and stirred at room temperature overnight. The reactionmixture was concentrated under reduced pressure, diluted with water andbrine and extracted three times with ethyl acetate. The combined organiclayers were washed twice with water and dried over sodium sulfate andfiltered. The solvent was removed under reduced pressure and crude14-O-{[(1R,2R)-2-(tert-butyl-diphenyl-silanyloxy)-4-hydroxyimino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer (quantitative yield, cyclohexane/ethyl acetate=2/1,R_(f)=0.25, 0.35) is obtained which was used for the next step withoutfurther purification.

Step E.14-O-{[(1R,2R,4S)-2-(tert-Butyl-diphenyl-silanyloxy)-4-hydroxyamino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R)diastereomer and14-O-{[(1R,2R,4R)-2-(tert-Butyl-diphenyl-silanyloxy)-4-hydroxyamino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S)diastereomer

14-O-{[(1R,2R)-2-(tert-Butyl-diphenyl-silanyloxy)-4-hydroxyimino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer (2.55 g, 3.36 mmol) was dissolved in 10 ml of acetic acidand treated with sodium cyanoborohydride (210 mg, 3.36 mmol) for 90minutes at room temperature. The reaction mixture was concentrated underreduced pressure. The residue was diluted with NaHCO₃ solution andextracted three times with ethyl acetate. The combined organic layerswere dried over sodium sulfate and filtered. The filtrate was submittedto chromatography (silica, cyclohexane/ethyl acetate=2/3) to yield14-O-{[(1R,2R,4S)-2-(tert-butyl-diphenyl-silanyloxy)-4-hydroxyamino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R)diastereomer (a) (R_(f)=0.5, 590 mg, 23% yield) and14-O-{[(1R,2R,4R)-2-(tert-butyl-diphenyl-silanyloxy)-4-hydroxyamino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S)diastereomer (b) (R_(f)=0.3, 670 mg, 26% yield).

(a): ¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 7.6-7.35 (m, 10H,aromat.-H), 6.93 (bs, 1H, NH/OH), 6.12, 6.08 (2dd, 1H, 19-H, J=11 Hz and18 Hz), 5.50 (m, 2H, 14-H, NH/OH), 5.00 (m, 2H, 20-H), 4.47 (m, 1H,11-OH), 3.95 (m, 1H, 2′-H), 3.40 (t, 1H, 11-H, J=6 Hz), 3.10-2.60 (m,4H, 1′-H, 4′-H, 22-H), 2.40 (bs, 1H, 4-H), 1.31, 1.30 (2s, 3H, 15-CH₃),1.00 (s, 12H, 18-CH₃, Si-tert-butyl), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.55(d, 3H, 16-CH₃, J=7 Hz).

(b): ¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 7.7-7.35 (m, 10H,aromat.-H), 6.85 (s, 1H, NH/OH), 6.16, 6.04 (2dd, 1H, 19-H, J=11 Hz and18 Hz), 5.55 (m, 2H, 14-H, NH/OH), 5.05 (m, 2H, 20-H), 4.49 (d, 1H,11-OH, J=6 Hz), 3.55 (m, 1H, 2′-H), 3.42 (t, 1H, 11-H, J=6 Hz),AB-system (ν_(A)=3.37, ν_(B)=3.18, 22-H, J=14 Hz), 2.88 (m, 1H, 1′-H),2.54 (m, 1H, 4′-H), 2.40 (bs, 1H, 4-H), 1.39, 1.37 (2s, 3H, 15-CH₃),1.06 (s, 3H, 18-CH₃), 1.00 (s, 9H, Si-tert-butyl), 0.83 (d, 3H, 17-CH₃,J=7 Hz), 0.64, 0.62 (2d, 3H, 16-CH₃, J=7 Hz).

Step F.14-O-{[(1R,2R,4S)-2-(tert-Butyl-diphenyl-silanyloxy)-4-(formyl-hydroxy-amino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R)diastereomer

To a solution of14-O-{[(1R,2R,4S)-2-(tert-butyl-diphenyl-silanyloxy)-4-hydroxyamino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R)diastereomer (474 mg, 0.622 mmol) in 15 ml of tert-butyl methyl etherwas added 2,2,2-trifluoroethyl formate (594 μl, 6.22 mmol) and heated toreflux for 5 hours. The reaction mixture was cooled to room temperatureand added dropwise to 150 ml of heptane. The resulting precipitate wasisolated by filtration to give14-O-{[(1R,2R,4S)-2-(tert-butyl-diphenyl-silanyloxy)-4-(formyl-hydroxy-amino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R)diastereomer (307 mg, 62% yield, cyclohexane/ethyl acetate=1/3,R_(f)=0.5) as colorless solid.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 9.6, 9.2 (2bs, 1H, NOH),8.2, 7.9 (2s, 1H, CHO), 7.65-7.35 (m, 10H, aromat.-H), 6.12, 6.08 (2dd,1H, 19-H, J=11 Hz and 18 Hz), 5.50 (d, 1H, 14-H, J=7 Hz), 5.05 (m, 2H,20-H), 4.47 (m, 1H, 11-OH), 3.40 (t, 1H, 11-H, J=6 Hz), 2.37 (bs, 1H,4-H), 1.31, 1.30 (2s, 3H, 15-CH₃), 1.03 (s, 12H, 18-CH₃, Si-tert-butyl),0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.55 (d, 3H, 16-CH₃, J=6 Hz).

Step G.14-O-{[(1R,2R,4S)-4-(Formyl-hydroxy-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R)diastereomer

14-O-{[(1R,2R,4S)-2-(tert-Butyl-diphenyl-silanyloxy)-4-(formyl-hydroxy-amino-cyclohexyl-sulfanyl]-acetyl}-mutilin+(1S,2S,4R)diastereomer (215 mg, 0.272 mmol) in 10 ml of tetrahydrofuran wastreated with tetrabutylammonium fluoride (1.36 ml, 1M in THF, 1.36 mmol)and stirred for 24 hours at room temperature. The reaction was dilutedwith a mixture of water, NaHCO₃ solution and brine (1:1:1) and extractedthree times with ethyl acetate. The combined organic layers were driedover sodium sulfate, filtered and concentrated under reduced pressure.The residue was added dropwise to 250 ml heptane. The resultingprecipitate was isolated by filtration to yield14-O-{[(1R,2R,4S)-4-(formyl-hydroxy-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R)diastereomer (97 mg, 65% yield, dichloromethane/methanol=9/1, R_(f)=0.4)as colorless solid.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 9.65, 9.25 (2bs, 1H,NOH), 8.2, 7.9 (2s, 1H, CHO), 6.13 (m, 1H, 19-H), 5.54 (d, 1H, 14-H, J=8Hz), 5.05 (m, 2H, 20-H), 4.49 (d, 1H, 11-OH, J=6 Hz), 3.42 (t, 1H, 11-H,J=6 Hz), 2.40 (bs, 1H, 4-H), 1.36 (s, 3H, 15-CH₃), 1.06 (s, 12H,18-CH₃), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.63 (d, 3H, 16-CH₃, J=6 Hz).MS-ESI (m/z): 574 (MNa⁺), 550 (M-H)⁻, 1101 (2M-H)⁻.

Example14—14-O-{[(1R,2R,5S)-5-(Formyl-hydroxy-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer Step A.(7R,8R)-8-(2,4,6-Trimethylbenzylsufanyl)-1,4-dioxa-spiro[4.5]decan-7-ol+(7S,8S)diastereomer

7,8-Epoxy-1,4-dioxa-spiro[4.5]decane (Zhang, L.; Koreeda, M. OrganicLetters 2002, 4(21), 3755-3758.) (22 g, 120 mmol) was treated with2,4,6-trimethylbenzyl mercaptan (20 g, 120 mmol) according to the methodof Example 1 Step A2. After work up and chromatography of the reactionmixture (silica, cyclohexane/ethyl acetate=2/1)(7R,8R)-8-(2,4,6-trimethylbenzylsufanyl)-1,4-dioxa-spiro[4.5]decan-7-ol+(7S,8S)diastereomer (R_(f)=0.4, 33 g, 85% yield) was obtained as oil.

MS-ESI (m/z): 345 (MNa⁺), 667 (2MNa⁺).

Step B. Acetic acid(7R,8R)-7-(2,4,6-trimethylbenzylsufanyl)-1,4-dioxa-spiro[4.5]dec-8-ylester+(7S,8S) diastereomer

To a solution of triphenylphosphine (26.5 g, 101 mmol) in 500 ml oftetrahydrofuran under argon atmosphere was added isopropylazodicarboxylate (19.6 ml, 101 mmol) and stirred for 30 minutes. Then(7R,8R)-8-(2,4,6-trimethylbenzylsufanyl)-1,4-dioxa-spiro[4.5]decan-7-ol+(7S,8S)diastereomer (27.7 g, 86 mmol) in 150 ml of tetrahydrofuran and aceticacid (7.7 ml, 135 mmol) were added and the reaction mixture was heatedto 80° C. for 24 hours. The resulting reaction mixture was concentratedunder reduced pressure and subjected to chromatography (silica,cyclohexane/ethyl acetate/methanol=3/1) to yield acetic acid(7R,8R)-7-(2,4,6-trimethylbenzylsufanyl)-1,4-dioxa-spiro[4.5]dec-8-ylester+(7S,8S) diastereomer (R_(f)=0.4, 7.0 g, 22% yield).

¹H NMR (500 MHz, CDCl₃, δ, ppm): 6.81 (s, 2H, aromat.-H), 4.85 (m, 1H,CHO), 3.96 (m, 4H, OCH₂CH₂O), AB-system (ν_(A)=3.83, ν_(B)=3.79, J=11Hz, SCH₂), 2.99 (m, 1H, CHS), 2.36 (s, 6H, CH₃), 2.23 (s, 3H, CH₃), 2.18(m, 1H), 2.12 (m, 1H), 2.11 (s, 3H, COCH₃), 1.90-1.58 (m, 4H). MS-ESI(m/z): 387 (MNa⁴).

Step C. (7R,8R)-7-Mercapto-1,4-dioxa-spiro[4.5]decan-8-ol+(7S,8S)diastereomer

Acetic acid(7R,8R)-7-(2,4,6-trimethylbenzylsufanyl)-1,4-dioxa-spiro[4.5]dec-8-ylester+(7S,8S) diastereomer (6.33 g, 17.4 mmol) was treated with sodium(1.6 g, 69.5 mmol) according to the method of Example 11 Step D. Afterwork up and chromatography of the reaction mixture (silica,cyclohexane/ethyl acetate=1/1)(7R,8R)-7-mercapto-1,4-dioxa-spiro[4.5]decan-8-ol+(7S,8S) diastereomer(R_(f)=0.4, 1.36 g, 38%) was obtained.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm): 4.89 (d, 1H, OH), 3.83 (m, 4H,OCH₂CH₂O), 3.17 (m, 1H, CHO), 2.76 (m, 1H, CHS), 2.43 (s, 1H, SH),1.90-1.30, 6H). MS-ESI (m/z): 189 (M-H)⁻.

Step D.14-O-{[(7R,8R)-8-Hydroxy-1,4-dioxa-spiro[4.5]dec-7-ylsulfanyl]-acetyl}-mutilin+(7S,8S)diastereomer

(7R,8R)-7-Mercapto-1,4-dioxa-spiro[4.5]decan-8-ol+(7S,8S) diastereomer(1.36 g, 7.15 mmol) was treated with pleuromutilin tosylate (3.8 g, 7.15mmol) according to the method of Example 11 Step E. After work up andchromatography of the reaction mixture (silica, cyclohexane/ethylacetate=1/1)14-O-{[(7R,8R)-8-hydroxy-1,4-dioxa-spiro[4.5]dec-7-ylsulfanyl]-acetyl}-mutilin+(7S,8S)diastereomer (R_(f)=0.25, 1.90 g, 48%) was obtained as colorlessamorphous foam.

MS-ESI(m/z): 573 (MNa⁺), 1123 (2MNa⁺), 549 (M-H)⁻, 585 (MCl⁻).

Step E.14-O-{[(7R,8R)-8-(tert-Butyl-diphenyl-silanyloxy)-1,4-dioxa-spiro[4.5]dec-7-ylsulfanyl]-acetyl}-mutilin+(7S,8S)diastereomer

14-O-{[(7R,8R)-8-Hydroxy-1,4-dioxa-spiro[4.5]dec-7-ylsulfanyl]-acetyl}-mutilin+(7S,8S)diastereomer (1.90 g, 3.45 mmol) was treated according to the method ofExample 13 Step B. After work up and chromatography of the reactionmixture (silica, cyclohexane/ethyl acetate=3/2)14-O-{[(7R,8R)-8-(tert-butyl-diphenyl-silanyloxy)-1,4-dioxa-spiro[4.5]dec-7-ylsulfanyl]-acetyl}-mutilin+(7S,8S)diastereomer (R_(f)=0.6, 1.65 g, 61%) was obtained as colorlessamorphous foam.

¹H NMR (400 MHz, DMSO-₆, δ, ppm, inter alia): 7.7-7.35 (m, 10H,aromat.-H), 6.13, 6.12 (2dd, 1H, 19-H, J=11 Hz and 18 Hz), 5.53 (d, 1H,14-H, J=7 Hz), 5.05 (m, 2H, 20-H), 4.50 (m, 1H, 11-OH), 3.78 (m, 4H,OCH₂CH₂O), 3.70 (m, 1H, 1′-H), 3.42 (m, 1H, 11-H), 3.05 (m, 3H, 2′-H,22-H), 2.40 (bs, 1H, 4-H), 1.36, 1.34 (2s, 3H, 15-CH₃), 1.05 (s, 3H,18-CH₃), 1.00 (s, 9H, Si-tert-butyl), 0.81 (d, 3H, 17-CH₃, J=7 Hz),0.60, 0.58 (2d, 3H, 16-CH₃, J=7 Hz).

Step F.14-O-{[(1R,2R)-2-(tert-Butyl-diphenyl-silanyloxy)-5-oxo-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer

14-O-{[(7R,8R)-8-(tert-Butyl-diphenyl-silanyloxy)-1,4-dioxa-spiro[4.5]dec-7-ylsulfanyl]-acetyl}-mutilin+(7S,8S)diastereomer (1.65 g, 2.09 mmol) was treated according to the method ofExample 13 Step C. Crude14-O-{[(1R,2R)-2-(tert-butyl-diphenyl-silanyloxy)-5-oxo-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer (1.34 g, 86% yield, cyclohexane/ethyl acetate=2/1,R_(f)=0.3) was obtained as colorless amorphous foam which was directlyused for the next step.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 7.7-7.35 (m, 10H,aromat.-H), 6.11, 6.09 (2dd, 1H, 19-H, J=11 Hz and 18 Hz), 5.48 (d, 1H,14-H, J=7 Hz), 4.98 (m, 2H, 20-H), 4.47 (m, 1H, 11-OH), 4.03 (m, 1H,1′-H), 3.45-2.95 (m, 4H, 11-H, 2′-H, 22-H), 2.37 (bs, 1H, 4-H), 1.31,1.29 (2s, 3H, 15-CH₃), 1.02 (s, 12H, 18-CH₃, Si-tert-butyl), 0.81 (d,3H, 17-CH₃, J=7 Hz), 0.53 (d, 3H, 16-CH₃, J=7 Hz). MS-ESI (m/z): 767(MNa³⁰ ), 779 (MCl⁻).

Step G.14-O-{[(1R,2R)-2-(tert-Butyl-diphenyl-silanyloxy)-5-hydroxyimino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer

14-O-{[(1R,2R)-2-(tert-Butyl-diphenyl-silanyloxy)-5-oxo-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer (1.34 g, 1.80 mmol) was treated according to the method ofExample 13 Step D. Crude14-O-{[(1R,2R)-2-(tert-butyl-diphenyl-silanyloxy)-5-hydroxyimino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer (quantitative yield, cyclohexane/ethyl acetate=1/1,R_(f)=0.6) was obtained as colorless amorphous foam which was directlyused for the next step.

Step H.14-O-{[(1R,2R,5S)-2-(tert-Butyl-diphenyl-silanyloxy)-5-hydroxyamino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer and14-O-{[(1R,2R,5R)-2-(tert-Butyl-diphenyl-silanyloxy)-5-hydroxyamino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5S)diastereomer

14-O-{[(1R,2R)-2-(tert-butyl-diphenyl-silanyloxy)-5-hydroxyimino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer (2.55 g, 3.36 mmol) was treated according to the method ofExample 13 Step E. After work up and chromatography of the reactionmixture (silica, cyclohexane/ethyl acetate=1/3)14-O-{[(1R,2R,5S)-2-(tert-butyl-diphenyl-silanyloxy)-5-hydroxyamino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (a) (R_(f)=0.4, 220 mg, 16% yield) and14-O-{[(1R,2R,5R)-2-(tert-butyl-diphenyl-silanyloxy)-5-hydroxyamino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5S)diastereomer (b) (R_(f)=0.25, 560 mg, 41% yield) were obtained.

(a): ¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 7.65-7.35 (m, 10H,aromat.-H), 7.00 (bs, 1H, NH/OH), 6.11, 6.09 (2dd, 1H, 19-H, J=11 Hz and18 Hz), 5.50 (d, 1H, 14-H, J=8 Hz), 5.00 (m, 2H, 20-H), 4.47 (m, 1H,11-OH), 3.80 (m, 1H, 2′-H), 3.40 (t, 1H, 11-H, J=6 Hz), 3.00 (m, 1H,1′-H), AB-system (ν_(A)=3.93, ν_(B)3.80, 22-H, J=15 Hz), 2.68 (m, 1H,5′-H), 2.40 (bs, 1H, 4H), 1.31, 1.29 (2s, 3H, 15-CH₃), 1.00 (s, 12H,18-CH₃, Si-tert-butyl), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.55 (d, 3H,16-CH₃, J=7 Hz).

(b): ¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 7.7-7.35 (m, 10H,aromat.-H), 6.97 (s, 1H, NH/OH), 6.16, 6.14 (2dd, 1H, 19-H, J=11 Hz and18 Hz), 5.56 (m, 2H, 14-H), 5.40 (bs, 1H, NH/OH), 5.07 (m, 2H, 20-H),4.49, 4.48 (2d, 1H, 11-OH, J=6 Hz), 3.48 (m, 1H, 2′-H), 3.43 (t, 1H,11-H, J=6 Hz), 3.24 (m, 2H, 22-H), 2.79 (m, 1H, 1′-H), 2.40 (bs, 1H,4-H), 2.33 (m, 1H, 5′-H), 1.38, 1.35 (2s, 3H, 15-CH₃), 1.05 (s, 3H,18-CH₃), 0.98 (s, 9H, Si-tert-butyl), 0.81 (d, 3H, 17-CH₃, J=7 Hz),0.63, 0.61 (2d, 3H, 16-CH₃, J=6 Hz).

Step I.14-O-{[(1R,2R,5S)-2-(tert-Butyl-diphenyl-silanyloxy)-5-(formyl-hydroxy-amino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer

14-O-{[(1R,2R,5S)-2-(tert-butyl-diphenyl-silanyloxy)-5-hydroxyamino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (215 mg, 0.282 mmol) was treated according to the method ofExample 13 Step F. Isolation of the precipitate by filtration resultedin14-O-{[(1R,2R,5S)-2-(tert-butyl-diphenyl-silanyloxy)-5-(formyl-hydroxy-amino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (135 mg, 61% yield, cyclohexane/ethyl acetate=1/3,R_(f)=0.65) as colorless solid.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 9.8, 9.3 (2bs, 1H, NOH),8.2, 7.9 (2bs, 1H, CHO), 7.60-7.35 (m, 10H, aromat.-H), 6.11, 6.09 (2dd,1H, 19-H, J=11 Hz and 18 Hz), 5.50 (d, 1H, 14-H, J=8 Hz), 5.00 (m, 2H,20-H), 4.47 (d, 1H, 11-OH, J=6 Hz), 3.40 (t, 1H, 11-H, J=6 Hz), 2.37(bs, 1H, 4-H), 1.32, 1.30 (2s, 3H, 15-CH₃), 1.03 (s, 12H, 18-CH₃,Si-tert-butyl), 0.82, 0.80 (d, 3H, 17-CH₃, J=7 Hz), 0.55 (d, 3H, 16-CH₃,J=6 Hz).

Step J.14-O-{[(1R,2R,5S)-5-(Formyl-hydroxy-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer

14-O-{[(1R,2R,5S)-2-(tert-Butyl-diphenyl-silanyloxy)-5-(formyl-hydroxy-amino-cyclohexyl-sulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (130 mg, 0.164 mmol) was treated according to the method ofExample 13 Step G. Isolation of the precipitate by filtration resultedin14-O-{[(1R,2R,5S)-5-(formyl-hydroxy-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (77 mg, 85% yield, dichloromethane/methanol=9/1, R_(f)=0.4)as colorless solid.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 9.7, 9.3 (2bs, 1H, NOH),8.2, 7.9 (2s, 1H, CHO), 6.13 (dd, 1H, 19-H, J=11 Hz and 18 Hz), 5.53 (d,1H, 14-H, J=8 Hz), 5.06 (m, 2H, 20-H), 4.91 (d, 1H, 2′-OH), 4.49 (d, 1H,11-OH, J=6 Hz), 4.2, 3.7 (2m, 2H, 2′-H, 5′-H), 3.41 (t, 1H, 11-H, J=6Hz), 3.28 (m, 2H, 22-H), 3.13 (m, 1H, 1′-H), 2.40 (bs, 1H, 4-H), 1.35(s, 3H, 15-CH₃), 1.06 (s, 12H, 18-CH₃), 0.81 (d, 3H, 17-CH₃, J=7 Hz),0.63 (d, 3H, 16-CH₃, J=6 Hz). MS-ESI (m/z): 574 (MNa⁺), 1125 (MNa⁺), 550(M-H)⁻, 1101 (2M-H)⁻.

Example15—14-O-{[(1R,2R,3R/S)-3-(Formyl-hydroxy-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3R/S)diastereomer Step A.14-O-{[(6R,7R)-6-Hydroxy-1,4-dioxa-spiro[4.5]dec-7-ylsulfanyl]-acetyl}-mutilin+(6S,7S)diastereomer

6,7-Epoxy-1,4-dioxa-spiro[4.5]decane (Vankar, Y. D.; Reddy M. V.;Chaudhuri, N. C. Tetrahedron 1994, 50(37), 11057-11078.) (16.24 g, 104mmol) and pleuromutilin thiol (20.5 g, 52 mmol) were treated accordingto the method of Example 1 Step A1. After work up and chromatography ofthe reaction mixture (silica, cyclohexane/dioxane=2/1)14-O-{[(6R,7R)-6-hydroxy-1,4-dioxa-spiro[4.5]dec-7-ylsulfanyl]-acetyl}-mutilin+(6S,7S)diastereomer (R_(f)=0.5, 15.6 g, 55% yield) was obtained as colorlessamorphous foam.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.13 (dd, 1H, 19-H, J=11Hz and 18 Hz), 5.53 (d, 1H, 14-H, J=7 Hz), 5.05 (m, 2H, 20-H), 4.90 (m,1H, 2′-OH), 4.47 (m, 1H, 11-OH), 3.97 (m, 1H, 2′-H), 3.32 (m, 1H, 11-H),3.50-3.20 (m, 2H, 22-H), 2.80 (m, 1H, 1′-H), 2.40 (bs, 1H, 4-H), 1.35,1.34 (2s, 3H, 15-CH₃), 1.05 (s, 3H, 18-CH₃), 0.81 (d, 3H, 17-CH₃, J=7Hz), 0.62 (d, 3H, 16-CH₃, J=6 Hz).

Step B.14-O-{[(1R,2R)-2-Hydroxy-3-oxo-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer

14-O-{[(6R,7R)-6-Hydroxy-1,4-dioxa-spiro[4.5]dec-7-ylsulfanyl]-acetyl}-mutilin+(6S,7S)diastereomer (15.6 g, 28.4 mmol) was treated according to the method ofExample 13 Step C. After work up and chromatography of the reactionmixture (silica, cyclohexane/ethyl acetate=1/1)14-O-{[(1R,2R)-2-hydroxy-3-oxo-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer (R_(f)0.4, 3.14 g, 22% yield) was obtained as colorlessamorphous foam.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.13 (dd, 1H, 19-H, J=11Hz and 18 Hz), 5.55 (d, 1H, 14-H, J=8 Hz), 5.23 (m, 1H, 2′-OH), 5.05 (m,2H, 20-H), 4.49 (d, 1H, 11-OH, J=6 Hz), 4.00 (m, 1H, 2′-H), 3.50-3.30(m, 3H, 11-H, 22-H), 2.86 (m, 1H, 1′-H), 2.40 (bs, 1H, 4-H), 1.35 (s,3H, 15-CH₃), 1.06 (s, 3H, 18-CH₃), 0.80 (d, 3H, 17-CH₃, J=7 Hz), 0.61(d, 3H, 16-CH₃, J=7 Hz). MS-ESI (m/z): 529 (MNa⁺), 1035 (2MNa⁺).

Step C.14-O-{[(1R,2R)-2-Hydro-3-hydroxyimino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer

14-O-{[(1R,2R)-2-Hydroxy-3-oxo-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer (3.14 g, 6.19 mmol) was treated according to the method ofExample 13 Step D. After work up and chromatography of the reactionmixture (silica, cyclohexane/ethyl acetate=1/1)14-O-{[(1R,2R)-2-hydroxy-3-hydroxyimino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer (R_(f)=0.2, 1.75 g, 54% yield) was obtained as colorlessamorphous foam.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 10.5 (s, 1H, NOH), 6.13,6.12 (2dd, 1H, 19-H, J=11 Hz and 18 Hz), 5.54 (d, 1H, 14-H, J=8 Hz),5.33 (d, 1H, 2′-OH, J=4 Hz), 5.05 (m, 2H, 20-H), 4.50 (m, 1H, 11-OH),3.96 (m, 1H, 2′-H), 3.42 (t, 1H, 11-H, J=6 Hz), 3.25 (m, 2H, 22-H), 3.14(m, 1H, 1′-H), 2.40 (bs, 1H, 4-H), 1.35 (s, 3H, 15-CH₃), 1.05 (s, 3H,18-CH₃), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.63 (d, 3H, 16-CH₃, J=7 Hz).

Step D.14-O-{[(1R,2R,3R/S)-2-Hydroxy-3-hydroxyamino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3R/S)diastereomer

14-O-{[(1R,2R)-2-Hydroxy-3-hydroxyimino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer (1.75 g, 3.35 mmol) was treated according to the method ofExample 13 Step E. After work up and chromatography of the reactionmixture (silica, ethyl acetate/methanol=10/1)14-O-{[(1R,2R,3R/S)-2-hydroxy-3-hydroxyamino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3R/S)diastereomer (R_(f)=0.2, 1.34 g, 65% yield) was obtained as colorlessamorphous foam.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 7.1 (bs, 1H, NH/OH),6.12, 6.11 (2dd, 1H, 19-H, J=11 Hz and 18 Hz), 5.53 (d, 1H, 14-H, J=8Hz), 5.05 (m, 2H, 20-H), 4.90 (m, 1H, 2′-OH), 4.5 (m, 1H, 11-OH), 3.41(t, 1H, 11-H, J=6 Hz), 3.73, 3.53, 3.30, 3.14, 3.01, 2.87 (6m, 5H, 1′-H,2′-H, 3′-H, 22-H), 2.40 (bs, 1H, 4-H), 1.35 (2s, 3H, 15-CH₃), 1.05 (s,3H, 18- CH₃), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.63 (d, 3H, 16-CH₃, J=7Hz).

Step E.14-O-{[(1R,2R,3R/S)-3-(Formyl-hydroxy-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3R/S)diastereomer

14-O-{[(1R,2R,3R/S)-2-hydroxy-3-hydroxyamino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3R/S)diastereomer (899 mg, 1.72 mmol) was treated according to the method ofExample 13 Step F. After isolation of the precipitate by filtration14-O-{[(1R,2R,3R/S)-3-(formyl-hydroxy-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,3R/S)diastereomer (724 mg, 76% yield, dichloromethane/methanol=9/1,R_(f)=0.5) was obtained as colorless solid.

1H NMR (400 MHz, DMSO-₆, δ, ppm, inter alia): 9.6, 9.9.4, 9.1 (3bs, 1H,NOH), 8.2, 7.9 (2s, 1H, CHO), 6.13, 6.11 (2dd, 1H, 19-H, J=11 Hz and 18Hz), 5.54 (d, 1H, 14-H, J=8 Hz), 5.24 (m, 1H, 2′-OH), 5.05 (m, 2H,20-H), 4.49 (m, 1H, 11-OH), 3.86, 3.60 (2m, 1H, 2′-H), 3.39 (t, 1H,11-H, J=6 Hz), 3.28, 3.13, 2.64 (3m, 4H, 1′-H, 3′-H, 22-H), 2.38 (bs,1H, 4-H), 1.36 (s, 3H, 15-CH₃), 1.06 (s, 12H, 18-CH₃), 0.81 (d, 3H,17-CH₃, J=7 Hz), 0.63 (d, 3H, 16- CH₃, J=7 Hz). MS-ESI (m/z): 574(MNa⁺), 1125 (2MNa⁺), 550 (M-H)⁻, 1101 (2M-H)⁻.

Example16—14-O-{[(1R,2R,5S)-2-Hydroxy-5-methylamino-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,5R) diastereomer hydrochloride Step A.N-Methyl-N-(cyclohex-3-enyl)-carbamic acid tert-butyl ester

Cyclohex-3-enyl-carbamic acid tert-butyl ester (Kampferer, P.; Vasella,A. Helvetica Chimica Acta 2004, 87, 2764-2789) (3 g, 15.2 mmol) andmethyl iodide (0.95 ml, 15.2 mmol) were treated for 1 hour according tothe method of Example 7 Step A. After work up and chromatography of thereaction mixture (silica, cyclohexane/ethyl acetate=10/1) the titlecompound (R_(f)=0.22, 2.04 g, 64% yield) was obtained as colorlesssolid.

¹H NMR (200 MHz, CDCl₃, δ, ppm): 5.64 (bs, 2H, double bond), 4.17 (bs,1H, NCH), 2.74 (s, 3H, NCH₃), 2.13, 1.70 (2m, 6H), 1.47 (s, 9H,tert-butyl).

Step B. N-Methyl-N-(cis-3,4-epoxycyclohexyl)-carbamic acid tert-butylester

N-Methyl-N-(cyclohex-3-enyl)-carbamic acid tert-butyl ester (2 g, 9.5mmol) and 3-chloroperbenzoic acid (2.2 g, 70%, 8.9 mmol) were treatedfor 1 hour according to the method of Example 7 Step B. After work upthe crude title compound (silica, cyclohexane/ethyl acetate=3/1,R_(f)=0.25, 1.70 g, 79% yield) was obtained.

¹H NMR (200 MHz, CDCl₃, δ, ppm): 4.0 (bs, 1H, NCH), 3.15 (bs, 2H,epoxide), 2.67 (s, 3H, NCH₃), 2.30-1.10 (m, 6H), 1.45 (s, 9H,tert-butyl).

Step C.14-O-{[(1R,2R,5S)-5-(tert-Butoxycarbonyl-methyl-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer

N-Methyl-N-(cis-3,4-epoxycyclohexyl)-carbamic acid tert-butyl ester (1.7g, 7.5 mmol) was treated with pleuromutilin thiol (2.95 g, 7.5 mmol)according to the method of Example 1 Step A3. After work up andchromatography of the reaction mixture (silica, cyclohexane/ethylacetate=2/1)14-O-{[(1R,2R,5S)-5-tert-Butoxycarbonyl-methyl-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (R_(f)=0.23, 1.3 g, 28% yield) was obtained as colorlesssolid.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.12 (2dd, 1H, 19-H, J=11Hz and 18 Hz), 5.54 (d, 1H, 14-H, J=8 Hz), 5.05 (m, 2H, 20-H), 4.9 (d,1H, 2′-OH, J=4 Hz), 4.47 (d, 1H, 11-OH, J=6 Hz), 3.97 (m, 1H, 5′-H),3.70 (bs, 1H, 2′-H), 3.42 (m, 1H, 11-H), 3.28 (m, 2H, 22-H), 3.11 (m,1H, 1′-H), 2.62 (s, 3H, NCH₃), 2.40 (bs, 1H, 4-H), 1.37 (s, 9H,tert-butyl), 1.35 (s, 3H, 15-CH₃), 1.04 (s, 3H, 18-CH₃), 0.81 (d, 3H,17-CH₃, J=7 Hz), 0.63 (d, 3H, 16-CH₃, J=7 Hz).

Step D.14-O-{[(1R,2R,5S)-2-Hydroxy-5-methylamino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer

14-O-{[(1R,2R,5S)-5-(tert-Butoxycarbonyl-methyl-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (1.3 g, 2.1 mmol) was treated according to the method ofExample 1 Step B. After work up and chromatography of the reactionmixture (silica, dichloromethane/methanol/i-propanol/water/aceticacid=80/20/6/3/2) with subsequent basic extraction14-O-{[(1R,2R,5S)-2-hydroxy-5-methylamino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (R_(f)=0.4, 690 mg, 63%) was obtained as colorless solid.

¹H NMR (500 MHz, DMSO-d₆, δ, ppm, inter alia): 6.14 (dd, 1H, 19-H, J=11Hz and 18 Hz), 5.54 (d, 1H, 14-H, J=8 Hz), 5.06 (m, 2H, 20-H), 4.81 (bs,1H, 2′-OH), 4.52 (d, 1H, 11-OH, J=6 Hz), 3.51 (m, 1H, 2′-H), 3.43 (m,1H, 11-H), 3.30 (m, 2H, 22-H), 3.00 (m, 1H, 1′-H), 2.63 (m, 1H, 5′-H),2.41 (bs, 1H, 4-H), 2.29 (s, 3H, NCH₃), 1.37 (s, 3H, 15-CH₃), 1.06 (s,3H, 18-CH₃), 0.82 (d, 3H, 17-CH₃, J=7 Hz), 0.64 (d, 3H, 16-CH₃, J=7 Hz).

Step E.14-O-{[(1R,2R,5S)-2-Hydroxy-5-methylamino-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,5R) diastereomer hydrochloride

14-O{-[(1R,2R,5S)-2-Hydroxy-5-methylamino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (690 mg, 1.32 mmol) was treated according to the method ofExample 1 Step C to obtain14-O-{[(1R,2R,5S)-2-hydroxy-5-methylamino-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,5R) diastereomer hydrochloride (731 mg,quantitative yield) as colorless solid.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 8.65 (bs, 2H, NH₂ ⁺),6.14 (2dd, 1H, 19-H, J=11 Hz and 18 Hz), 5.54 (d, 1H, 14-H, J=8 Hz),5.06 (m, 2H, 20-H), 4.51 (d, 1H, 11-OH, J=6 Hz), 3.72 (m, 1H, 2′-H),3.43 (m, 1H, 11-H), 3.37-3.00 (m, 4H, 22-H, 1′-H, 5′-H), 2.50 (s, 3H,NCH₃), 2.40 (bs, 1H, 4-H), 1.36 (s, 3H, 15-CH₃), 1.06 (s, 3H, 18-CH₃),0.82 (d, 3H, 17-CH₃, J=7 Hz), 0.63 (d, 3H, 16-CH₃, J=7 Hz). MS-ESI(m/z): 522 (MH⁺), 556 (MCl⁻).

Example17—14-O-{[(1R,2R,5S)-5-Allylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,5R) diastereomer hydrochloride Step A.N-Allyl-N-(cyclohex-3-enyl)-carbamic acid tert-butyl ester

Cyclohex-3-enyl-carbamic acid tert-butyl ester (Kampferer, P.; Vasella,A. Helvetica Chimica Acta 2004, 87, 2764-2789) (3 g, 15.2 mmol) andallyl iodide (1.4 ml, 15.2 mmol) were treated overnight according to themethod of Example 7 Step A. After work up and chromatography of thereaction mixture (silica, cyclohexane/ethyl acetate=10/1) the titlecompound (R_(f)=0.55, 2.0 g, 55% yield) was obtained as colorless solid.

¹H NMR (200 MHz, CDCl₃, δ, ppm): 5.60 (m, 2H, double bond), 5.80, 5.10,3.64 (3m, 5H, allyl), 4.18 (bs, 1H, NCH), 2.14, 1.74 (2m, 6H), 1.45 (s,9H, tert-butyl).

Step B. N-Allyl-N-(cis-3,4-epoxycyclohexyl)-carbamic acid tert-butylester

N-Allyl-N-(cyclohex-3-enyl)-carbamic acid tert-butyl ester (2 g, 8.4mmol) and 3-chloroperbenzoic acid (2.2 g, 70%, 8.9 mmol) were treatedovernight according to the method of Example 7 Step B. After work up thecrude title compound (silica, cyclohexane/ethyl acetate=3/1, R_(f)=0.31,1.90 g, 89% yield) was obtained.

¹H NMR (200 MHz, CDCl₃, δ, ppm): 5.76, 5.10, 3.66 (3m, 5H, allyl), 4.04(bs, 1H, NCH), 3.12 (bs, 2H, epoxide), 2.30-1.20 (m, 6H), 1.47 (s, 9H,tert-butyl).

Step C.14-O-{[(1R,2R,5S)-5-(tert-Butoxycarbonyl-allyl-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer

N-Allyl-N-(cis-3,4-epoxycyclohexyl)-carbamic acid tert-butyl ester (1.9g, 7.5 mmol) was treated with Pleuromutilin thiol (2.95 g, 7.5 mmol)according to the method of Example 1 Step A2. After work up andchromatography of the reaction (silica, cyclohexane/ethylacetate=3/1−>1/1) a mixture of14-O-{[(1R,2R,5S)-5-(tert-Butoxycarbonyl-allyl-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer and Pleuromutilin disulfide (cyclohexane/ethyl acetate=1/1,R_(f)=0.21, 2.49 g) was obtained.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.12 (2dd, 1H, 19-H, J=11Hz and 18 Hz), 5.74 (m, 1H, NCH₂CHCH₂), 5.54 (d, 1H, 14-H, J=8 Hz), 5.05(m, 4H, 20-H, NCH₂CHCH₂), 4.87 (d, 1H, 2′-OH, J=3 Hz), 4.49 (d, 1H,11-OH, J=6 Hz), 3.95 (m, 1H, 5′-H), 3.68 (bs, 3H, 2′-H, NCH₂CHCH₂), 3.42(t, 1H, 11-H, J=6 Hz), 3.26 (m, 2H, 22-H), 3.09 (m, 1H, 1′-H), 2.39 (bs,1H, 4-H), 1.37 (s, 9H, tert-butyl), 1.35 (s, 3H, 15-CH₃), 1.05 (s, 3H,18-CH₃), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.63 (d, 3H, 16-CH₃, J=7 Hz).MS-ESI (m/z): 670 (MNa⁺), 1317 (2MNa⁺), 646 (M-H)⁻.

Step D.14-O-{[(1R,2R,5S)-5-Allylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer

The mixture of14-O-{[(1R,2R,5S)-5-(tert-Butoxycarbonyl-allyl-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer and Pleuromutilin disulfide (2.4 g) was treated accordingto the method of Example 1 Step B. After work up and chromatography ofthe reaction mixture (silica,dichloromethane/methanol/i-propanol/water/acetic acid=80/20/6/3/2) withsubsequent basic extraction14-O-{[(1R,2R,5S)-5-allylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (R_(f)=0.5, 250 mg) was obtained as colorless solid.

¹H NMR (500 MHz, DMSO-d₆, δ, ppm, inter alia): 6.14 (dd, 1H, 19-H, J=11Hz and 18 Hz), 5.82 (m, 1H, NCH₂CHCH₂), 5.55 (d, 1H, 14-H, J=8 Hz), 5.10(m, 4H, 20-H, NCH₂CHCH₂), 4.77 (m, 1H, 2′-OH), 4.51 (d, 1H, 11-OH, J=6Hz), 3.50-3.10 (m, 6H, 2′-H, 11-H, 22-H, NCH₂CHCH₂), 2.99 (m, 1H, 1′-H),2.68 (m, 1H, 5′-H), 2.40 (bs, 1H, 4-H), 1.37 (s, 3H, 15-CH₃), 1.06 (s,3H, 18-CH₃), 0.82 (d, 3H, 17-CH₃, J=7 Hz), 0.63 (d, 3H, 16-CH₃, J=7 Hz).MS-ESI (m/z): 548 (MH⁺), 546 (M-H)⁻, 582 (MCl⁻).

Step E.14-O-{[(1R,2R,5S)-5-Allylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,5R) diastereomer hydrochloride

14-O-{[(1R,2R,5S)-5-Allylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (250 mg, 0.46 mmol) was treated according to the method ofExample 1 Step C to obtain14-O-{[(1R,2R,5S)-2-hydroxy-5-ethylamino-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,5R) diastereomer hydrochloride (273 mg, quant.yield uncorrected) as colorless solid.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 8.85 (bs, 2H, NH₂ ⁺),6.13 (2dd, 1H, 19-H, J=11 Hz and 18 Hz), 5.87 (m, 1H, NCH₂CHCH₂), 5.55(d, 1H, 14-H, J=8 Hz), 5.47, 5.37 (2d, 2H, NCH₂CHCH₂, J=17 Hz and 10Hz), 5.06 (m, 2H, 20-H), 4.51 (d, 1H, 11-OH, J=6 Hz), 3.72 (m, 1H,2′-H), 3.56 (d, 2H, NCH₂CHCH₂, J=6 Hz), 3.43 (t, 1H, 11-H, J=6 Hz), 3.34(m, 2H, 22-H), 3.13 (m, 2H, 1′-H, 5′-H), 2.40 (bs, 1H, 4-H), 1.36 (s,3H, 15-CH₃), 1.06 (s, 3H, 18-CH₃), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.63(d, 3H, 16-CH₃, J=7 Hz). MS-ESI (m/z): 548 (MH⁺), 582 (MCl⁻).

Example18—14-O-{[(1R,2R,5S)-2-Hydroxy-5-(2-methoxy-ethylamino)-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,5R) diastereomer hydrochloride Step A.N-(2-methoxy-ethyl)-N-(cyclohex-3-enyl)-carbarmic acid tert-butyl ester

Cyclohex-3-enyl-carbamic acid tert-butyl ester (Kampferer, P.; Vasella,A. Helvetica Chimica Acta 2004, 87, 2764-2789) (3 g, 15.2 mmol) and2-bromoethyl methyl ether (1.43 ml, 15.2 mmol) were treated overnight tothe method of Example 7 Step A. After work up and chromatography of thereaction mixture (silica, cyclohexane/ethyl acetate=7/1) the titlecompound (R_(f)=0.33, 1.2 g, 31% yield) was obtained as colorless solid.

¹H NMR (200 MHz, CDCl₃, δ, ppm); 5.61 (m, 2H, double bond), 4.10 (bs,1H, NCH), 3.50-3.15 (m, 7H, NCH₂CH₂OCH₃), 2.15, 1.72 (2m, 6H), 1.47 (s,9H, tert-butyl).

Step B. N-(2-Methoxy-ethyl)-N-(cis-3,4-epoxycyclohexyl)-carbamic acidtert-butyl ester

N-(2-Methoxy-ethyl)-N-(cyclohex-3-enyl)-carbamic acid tert-butyl ester(1.2 g, 4.7 mmol) and 3-chloroperbenzoic acid (1.2 g, 70%, 4.87 mmol)were treated over the weekend according to the method of Example 7 StepB. After work up the crude title compound (silica, cyclohexane/ethylacetate=3/1, R_(f)=0.33, 1.08 g, 85% yield) was obtained.

¹H NMR (200 MHz, CDCl₃, δ, ppm): 3.94 (bs, 1H, NCH), 3.50-3.05 (m, 9H,NCH₂CH₂OCH₃, epoxide), 2.30-1.20 (m, 6H), 1.45 (s, 9H, tert-butyl).

Step C.14-O-{[(1R,2R,5S)-5-(tert-Butoxycarbonyl-(2-methoxy-ethyl)-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer

N-(2-Methoxy-ethyl)-N-(cis-3,4-epoxycyclohexyl)-carbamic acid tert-butylester (1.08 g, 4.0 mmol) was treated with pleuromutilin thiol (1.57 g,4.0 mmol) according to the method of Example 1 Step A2. After work upand chromatography of the reaction mixture (silica, cyclohexane/ethylacetate=1/2)14-O-{[(1R,2R,5S)-5-(tert-Butoxycarbonyl-(2-methoxy-ethyl)amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (R_(f)=0.5, 500 mg, 19% yield) was obtained as colorlesssolid.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.13 (2dd, 1H, 19-H, J=11Hz and 18 Hz), 5.54 (d, 1H, 14-H, J=8 Hz), 5.05 (m, 2H, 20-H), 4.88 (d,1H, 2′-OH, J=4 Hz), 4.48 (d, 1H, 11-OH, J=6 Hz), 3.85 (m, 1H, 5′-H),3.68 (bs, 1H, 2′-H), 3.42 (t, 1H, 11-H, J=6 Hz), 3.35-3.05 (m, 10H,22-H, NCH₂CH₂OCH₃, 1′-H), 2.40 (bs, 1H, 4-H), 1.38 (s, 9H, tert-butyl),1.35 (s, 3H, 15-CH₃), 1.05 (s, 3H, 18-CH₃), 0.81 (d, 3H, 17-CH₃, J=7Hz), 0.63 (d, 3H, 16-CH₃, J=7 Hz).

Step D.14-O-{[(1R,2R,5S)-2-Hydroxy-5-(2-methoxy-ethylamino)-cyclohexylsurfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer

14-O-{[(1R,2R,5S)-5-(tert-Butoxycarbonyl-(2-methoxyethyl)-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (500 mg, 0.75 mmol) was treated according to the method ofExample 1 Step B. After work up and chromatography of the reactionmixture (silica, dichloromethane/methanol/i-propanol/water/aceticacid=80/20/6/3/2) with subsequent basic extraction14-O-{[(1R,2R,5S)-2-hydroxy-5-(2-methoxy-ethylamino)-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (R_(f)=0.6, 330 mg, 78%) was obtained as colorless solid.

¹H NMR (500 MHz, DMSO-d₆, δ, ppm, inter alia): 6.14 (dd, 1H, 19-H, J=11Hz and 18 Hz), 5.54 (d, 1H, 14-H, J=8 Hz), 5.06 (m, 2H, 20-H), 4.74 (m,1H, 2′-OH), 4.51 (2d, 1H, 11-OH, J=6 Hz), 3.50-3.20 (m, 9H, 11-H, 2′-H,22-H, NCH₂CH₂OCH₃), 2.97 (m, 1H, 1′-H), 2.63 (m, 3H, 5′-H, NCH₂CH₂OCH₃),2.40 (bs, 1H, 4-H), 1.37 (s, 3H, 15-CH₃), 1.06 (s, 3H, 18-CH₃), 0.82 (d,3H, 17-CH₃, J=7 Hz), 0.63 (d, 3H, 16-CH₃, J=7 Hz).

Step E.14-O-{[(1R,2R,5S)-2-Hydroxy-5-(2-methoxy-ethylamino)-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,5R) diastereomer hydrochloride

14-O-{[(1R,2R,5S)-2-Hydroxy-5-(2-methoxy-ethylamino)-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (330 mg, 0.58 mmol) was treated according to the method ofExample 1 Step C to obtain14-O-{[(1R,2R,5S)-2-hydroxy-5-(2-methoxy-ethylamino)-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,5R) diastereomer hydrochloride (355 mg, quant.yield, uncorrected) as colorless solid.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 8.65 (bs, 2H, NH₂ ⁺),6.13 (2dd, 1H, 19-H, J=11 Hz and 18 Hz), 5.55 (d, 1H, 14-H, J=8 Hz),5.06 (m, 2H, 20-H), 4.51 (d, 1H, 11-OH, J=6 Hz), 3.71 (m, 1H, 2′-H),3.57 (m, 5H, NCH₂CH₂OCH₃), 3.42 (t, 1H, 11-H, J=6 Hz), 3.33 (m, 2H,22-H), 3.20-3.00 (m, 4H, 1′-H, 5′-H, NCH₂CH₂OCH₃), 2.40 (bs, 1H, 4-H),1.36 (s, 3H, 15-CH₃), 1.05 (s, 3H, 18-CH₃), 0.81 (d, 3H, 17-CH₃, J=7Hz), 0.63 (2d, 3H, 16-CH₃, J=7 Hz). MS-ESI (m/z): 566 (MH⁺), 600 (MCl⁻).

Example19—14-O-{[(1R,2R,4R*)-2-Hydroxy-4-(2-hydroxy-ethylamino)-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,4S*) diastereomer hydrochloride Step A.14-O-{[(1R,2R,4R/S)-2-(tert-Butyl-diphenyl-silanyloxy)-4-(2-hydroxy-ethylamino)-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R/S)diastereomer

To a solution of14-O-{[(1R,2R)-2-(tert-butyl-diphenyl-silanyloxy)-4-oxo-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer (1.50 g, 2.01 mmol) from Example 13 Step C in 20 ml ofdichloromethane was added ethanolamine (0.12 ml, 2.01 mmol) andtitanium(IV)isopropoxide (0.7 ml, 2.52 mmol) and stirred for 2 hours atroom temperature. The resulting reaction mixture was treated with sodiumcyanoborohydride (126 mg, 2 mmol) overnight at room temperature, dilutedwith further dichloromethane and extracted with NaHCO₃ solution. Theorganic layer was dried over sodium sulphate and filtered. The filtratewas subjected to chromatography (silica, dichloromethane/methanol=30/1)to yield14-O-{[(1R,2R,4R/S)-2-(tert-butyl-diphenyl-silanyloxy)-4-(2-hydroxy-ethylamino)-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R/S)diastereomer (R_(f)=0.3, 230 mg, 14% yield).

¹H NMR (500 MHz, CDCl₃, δ, ppm, inter alia): 7.8-7.30 (m, 10H,aromat.-H), 6.47 (m, 1H, 19-H), 5.75, 5.69 (2d, 1H, 14-H, J=8 Hz),5.40-5.15 (m, 1H, 20-H), 4.04, 3.65 (2m, 1H, 2′-H), 3.64, 3.51 (2m, 2H,NCH₂CH₂OH), 3.36 (m, 1H, 11-H), 2.74, 2.54 (2m, 2H, NCH₂CH₂OH), 2.11(bs, 1H, 4-H), 1.44, 1.45 (2s, 3H, 15-CH₃), 1.17, 1.16 (s, 3-H, 18-CH₃),1.08 (s, 9H, Si-tert-butyl), 0.88 (2d, 3H, 17-CH₃), 0.75-0.65 (m, 3H,16-CH₃). MS-ESI (m/z): 790 (MH⁺), 824 (MCl⁻).

Step B.14-O-{[(1R,2R,4R*)-2-Hydroxy-4-(2-hydroxy-ethylamino)-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4*S)diastereomer

To a solution of14-O-{[(1R,2R,4R/S)-2-(tert-butyl-diphenyl-silanyloxy)-4-(2-hydroxy-ethylamino)-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R/S)diastereomer (230 mg, 0.29 mmol) in 15 ml of acetonitrile was treatedwith HF (40% aqueous, 2 drops) and stirred overnight at roomtemperature. The reaction was charged with NaHCO₃ solution and extractedwith dichloromethane. The combined organic layers were dried over sodiumsulfate, filtered and concentrated under reduced pressure. Afterchromatography (silica, dichloromethane/methanol=6/1) the title compound(R_(f)=0.4, 50 mg, 31% yield) was obtained.

¹H NMR (400 MHz, CDCl₃, δ, ppm, inter alia): 6.48 (dd, 1H, 19-H, J=11 Hzand 17 Hz), 5.76, 5.68 (2d, 1H, 14-H, J=8 Hz), 5.40-5.15 (m, 1H, 20-H),3.66 (t, 2H, NCH₂CH₂OH, J=5 Hz), 3.50-3.15 (m, 4H, 2′-H, 11-H, 22H),2.80 (m, 2H, NCH₂CH₂OH), 2.63 (m, 2H, 1′-H, 4′-H), 2.11 (bs, 1H, 4-H),1.46 (s, 3H, 15-CH₃), 1.18 (s, 3H, 18-CH₃), 0.89 (d, 3H, 17-CH₃), 0.73(2d, 3H, 16-CH₃). MS-ESI (m/z): 552 (MH⁺).

Step C.14-O-{[(1R,2R,4R*)-2-Hydroxy-4-(2-hydroxy-ethylamino)-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,4S*) diastereomer hydrochloride

14-O-{[(1R,2R,4R*)-2-Hydroxy-4-(2-hydroxy-ethylamino)-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S*)diastereomer (50 mg, 0.091 mmol) was treated according to the method ofExample 1 Step C to obtain14-O-{[(1R,2R,4R*)-2-Hydroxy-4-(2-hydroxy-ethylamino)-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,4S*) diastereomer hydrochloride (43 mg, 80% yield)as colorless solid.

¹H NMR (500 MHz, CDCl₃, δ, ppm, inter alia): 6.46 (dd, 1H, 19-H, J=11 Hzand 17 Hz), 5.76 (d, 1H, 14-H, J=8 Hz), 5.37 (d, 1H, 20-H, J=11 Hz),5.23 (d, 1H, 20-H, J=17 Hz), 3.95 (m, 2H, NCH₂CH₂OH, J=5 Hz), 3.58 (m,1H, 2′-H), 3.40-3.10 (m, 5H, 11-H, 22H, NCH₂CH₂OH), 2.72 (m, 1H, 1′-H),2.11 (bs, 1H, 4-H), 1.46 (s, 3H, 15-CH₃), 1.18 (s, 3H, 18-CH₃), 0.89 (d,3H, 17-CH₃, J=7 Hz), 0.73 (d, 3H, 16-CH₃, J=7 Hz). MS-ESI (m/z): 552(MH⁺), 586 (MCl⁻).

Example20—14-O-{[(1R,2R,4R*)-4-Cyclohexylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,4S*) diastereomer hydrochloride Step A.14-O-{[(1R,2R,4R*)-2-(tert-Butyl-diphenyl-silanyloxy)-4-(2-hydroxy-ethylamino)-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S*)diastereomer

14-O-{[(1R,2R)-2-(tert-butyl-diphenyl-silanyloxy)-4-oxo-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer (1.50 g, 2.01 mmol) from Example 13 Step C was reacted withcyclohexylamine (0.23 ml, 1.01 mmol) according to the method of Example19 Step A. After work up and chromatography of the reaction mixture(silica, dichloromethane/methanol=30/1)14-O-{[(1R,2R,4R*)-2-(tert-butyl-diphenyl-silanyloxy)-4-cyclohexylamino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S*)diastereomer (R_(f)=0.13, 150 mg, 9% yield) was obtained.

¹H NMR (500 MHz, CDCl₃, δ, ppm, inter alia): 7.8-7.3 (m, 10H,aromat.-H), 6.48 (dd, 1H, 19-H, J=11 Hz and 17 Hz), 5.76 (d, 1H, 14-H,J=8 Hz), 5.40-5.15 (m, 1H, 20-H), 3.62 (m, 1H, 2′-H), 3.40-3.10 (m, 3H,11-H, 22-H), 2.77 (m, 1H, 1′-H), 2.11 (bs, 1H, 4-H), 1.46 (s, 3H,15-CH₃), 1.16 (s, 3H, 18-CH₃), 1.07 (s, 9H, Si-tert-butyl), 0.88 (2d,3H, 17-CH₃), 0.74, 0.73 (2d, 3H, 16-CH₃). MS-ESI (m/z): 828 (MH⁺), 862(MCl⁻).

Step B.14-O-{[(1R,2R,4R*)-4-Cyclohexyl-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S*)diastereomer

14-O-{[(1R,2R,4R*)-2-(tert-butyl-diphenyl-silanyloxy)-4-cyclohexylamino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S*)diastereomer (100 mg, 0.121 mmol) was treated with HF (40% aqueous, 30drops) for 5 hours according to the method of Example 19 Step B. Afterwork up and chromatography of the reaction mixture (silica,dichloromethane/methanol=10/1) the title compound (R_(f)=0.13, 23 mg,32% yield) was obtained.

¹H NMR (500 MHz, CDCl₃, δ, ppm, inter alia): 6.46 (dd, 1H, 19-H, J=11 Hzand 17 Hz), 5.75 (m, 1H, 14-H), 5.40-5.15 (m, 1H, 20H), 3.49 (m, 1H,2′-H), 3.35 (m, 1H, 11-H), AB-system (ν_(A)=3.30, ν_(B)=3.20, 22-H, J=15Hz), 3.01 (m, 1H, 4′-H), 2.67 (m, 1H, 1′-H) 2.09 (bs, 1H, 4-H), 1.45 (s,3H, 15-CH₃), 1.16 (s, 3H, 18-CH₃), 0.87 (d, 3H, 17-CH₃, J=7 Hz), 0.72(d, 3H, 16-CH₃, J=7 Hz). MS-ESI (m/z): 591 (MH⁺).

Step C.14-O-{[(1R,2R,4R*)-4-Cyclohexyl-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,4S*) diastereomer hydrochloride

14-O-{[(1R,2R,4R*)-4-Cyclohexyl-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S*)diastereomer (23 mg, 0.039 mmol) was treated according to the method ofExample 1 Step C to obtain14-O-{[(1R,2R,4R*)-4-Cyclohexyl-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,4S*) diastereomer hydrochloride (26 mg,quantitative yield, uncorrected) as colorless solid.

¹H NMR (500 MHz, CDCl₃, δ, ppm, inter alia): 9.3 (bs, 2H, NH₂ ⁺), 6.46(dd, 1H, 19-H, J=11 Hz and 17 Hz), 5.75 (d, 1H, 14-H, J=8 Hz), 5.38 (d,1H, 20-H, J=12 Hz), 5.22 (d, 1H, 20-H, J=17 Hz), 3.50-3.00 (m, 6H, 2′-H,11-H, 22H, NcHex), 2.65 (m, 2H, 1′-H, 3a′-H), 2.10 (bs, 1H, 4-H), 1.45(s, 3H, 15-CH₃), 1.18 (s, 3H, 18-CH₃), 0.88 (d, 3H, 17-CH₃, J=7 Hz),0.73 (d, 3H, 16-CH₃, J=7 Hz). MS-ESI (m/z): 591 (MH⁺), 624 (MCl⁻).

Example21—14-O-{[(1R,2R,4R*)-4-Cyclopropylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S*)diastereomer Step A.14-O-{[(1R,2R,4R*)-2-(tert-Butyl-diphenyl-silanyloxy)-4-cyclopropylamino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S*)diastereomer

14-O-{[(1R,2R)-2-(tert-butyl-diphenyl-silanyloxy)-4-oxo-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer (750 mg, 1.01 mmol) from Example 13 Step C was reacted withcyclopropylamine (0.07 ml, 1.01 mmol) in 40 ml of dichloromethaneaccording to the method of Example 19 Step A. After the treatment ofsodium cyanoborohydride ethanol was added (0.7 ml) and the mixture wasstirred overnight at room temperature. After work up and chromatographyof the reaction mixture (silica, dichloromethane/methanol=30/1)14-O-{[(1R,2R,4R*)-2-(tert-butyl-diphenyl-silanyloxy)-4-cyclopropylamino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S*)diastereomer (R_(f)=0.35, 283 mg, 36% yield) was obtained.

¹H NMR (500 MHz, DMSO-d₆, δ, ppm, inter alia): 7.65-7.35 (m, 10H,aromat.-H), 6.12 (m, 1H, 19-H), 5.5.2, 5.51 (2d, 1H, 14-H, J=8 Hz), 5.00(m, 1H, 20-H), 4.50 (t, 1H, 11-OH, J=5.5 Hz), 3.94 (m, 1H, 2′-H), 3.41(m, 1H, 11-H), 3.05-2.80 (m, 4H, 22-H, 1′-H, 4′-H), 2.39 (bs, 1H, 4-H),1.86 (m, 1H, cPr), 1.13 (2s, 3H, 15-CH₃), 1.12 (s, 12H, 18-CH₃,Si-tert-butyl), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.57, 0.56 (2d, 3H,16-CH₃, J=7 Hz), 0.25, 0.06 (2m, 4H, cPr). MS-ESI (m/z): 786 (MH⁺), 784(M-H)⁻.

Step B.14-O-{[(1R,2R,4R*)-4-Cyclopropylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S*)diastereomer

14-O-{[(1R,2R,4R*)-2-(tert-butyl-diphenyl-silanyloxy)-4-cyclopropyl-amino-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4S*)diastereomer (223 mg, 0.284 mmol) was treated overnight withtetrabutylammonium fluoride according to the method of Example 13 StepG. After work up and chromatography of the reaction mixture (silica,dichloromethane/methanol=10/1) the title compound (R_(f)=0.2, 10 mg, 6%yield) was obtained.

¹H NMR (500 MHz, DMSO-d₆, δ, ppm, inter alia): 6.13 (dd, 1H, 19-H, J=11Hz and 17 Hz), 5.50 (d, 1H, 14-H, J=7 Hz), 5.05 (m, 1H, 20-H), 4.87 (m,1H, 2′-OH), 4.50 (d, 1H, 11-OH, J=6 Hz), 3.55-3.20 (m, 4H, 22-H, 2′-H,11-H), 2.50 (m, 2H, 1′-H, 4′-H), 2.40 (bs, 1H, 4-H), 2.01 (m, 1H, cPr),1.35 (s, 3H, 15-CH₃), 1.04 (s, 3H, 18-CH₃), 0.81 (d, 3H, 17-CH₃, J=7Hz), 0.62 (d, 3H, 16-CH₃, J=6 Hz), 0.32, 0.15 (2m, 4H, cPr). MS-ESI(m/z): 548 (MH⁺), 1095 (2MH⁺), 1117 (2MNa⁺), 582 (MCl⁻).

Example22—14-O-{[(1R,2R,5S*)-4-Cyclopropylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,5R*) diastereomer hydrochloride Step A.14-O-{[(1R,2R)-2-Hydroxy-4-oxo-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer and14-O-{[(1R,2R)-2-Hydroxy-5-oxo-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer

14-O-{[(7R,8R)-7-hydroxy-1,4-dioxa-spiro[4.5]dec-8-ylsulfanyl]-acetyl}-mutilin+(7S,8S)diastereomer as well as14-O-{[(7R,8R)-8-hydroxy-1,4-dioxa-spiro[4.5]dec-7-ylsulfanyl]-acetyl}-mutilin+(7S,8S)diastereomer (3.96 g, 7.19 mmol) was dissolved in 50 ml of dioxane andtreated with 4N HCl (5 ml, 20 mmol) for 6 hours at room temperature. Thereaction mixture was concentrated under reduced pressure, charged withNaHCO₃ solution and extracted three times with ethyl acetate. Theorganic layers were dried over sodium sulphate and filtered. Thefiltrate was concentrated under reduced pressure and subjected tochromatography (silica, cyclohexane/dioxane=2/1) to yield a mixture ofthe title compounds (R_(f)=0.30, 860 mg, 24% yield).

¹H NMR (500 MHz, DMSO-d₆, δ, ppm, inter alia): 6.13 (m, 1H, 19-H), 5.56,5.54 (2d, 1H, 14-H, J=8 Hz), 5.38, 5.32 (2m, 1H, 2′-OH), 5.05 (m, 2H,20-H), 4.50 (d, 1H, 11-OH, J=5 Hz), 3.95, 3.83 (2m, 1H, 2′-H), 3.50-3.20(m, 3H, 11-H, 22-H), 3.17, 3.07 (2m, 1H, 1′-H), 2.40 (bs, 1H, 4-H),1.35, 1.33 (2s, 3H, 15-CH₃), 1.05 (s, 3H, 18-CH₃), 0.81 (d, 3H, 17-CH₃,J=7 Hz), 0.63, 0.62 (2d, 3H, 16-CH₃). 529 (MNa⁺), 505 (M-H)⁻.

Step B.14-O-{[(1R,2R,5S*)-5-Cyclopropylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R*)diastereomer and14-O-{[(1R,2R,4S*)-4-Cyclopropylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R*)diastereomer

14-O-{[(1R,2R)-2-Hydroxy-4-oxo-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer and14-O-{[(1R,2R)-2-hydroxy-5-oxo-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer (250 mg, 0.493 mmol) was reacted with cyclopropylamine(0.03 ml, 0.493 mmol) in 15 ml of dichloromethane according to themethod of Example 19 Step A. After the treatment of sodiumcyanoborohydride ethanol was added (0.7 ml) and the mixture was stirredovernight at room temperature. After work up and chromatography of thereaction mixture (silica, dichloromethane/methanol=20/1)14-O-{[(1R,2R,5S*)-4-cyclopropylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R*)diastereomer (a) (dichloromethane/methanol=10/1, R_(f)=0.22, 34 mg, 13%yield) and14-O-{[(1R,2R,4S*)-4-cyclopropylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R*)diastereomer (b) (dichloromethane/methanol=10/1, R_(f)=0.13, 26 mg, 4%yield) were obtained.

(a): ¹H NMR (500 MHz, DMSO-d₆, δ, ppm, inter alia): 6.13 (m, 1H, 19-H,J=11 and 18 Hz), 5.53 (d, 1H, 14-H, J=8 Hz), 5.06 (m, 1H, 20-H), 4.67(t, 1H, 2′-OH), 4.47 (d, 1H, 11-OH, J=6 Hz), 3.50-3.20 (m, 4H, 11-H,2′-H, 22-H) 2.92 (m, 1H, 1′-H), 2.71 (m, 1H, 5′-H), 2.39 (bs, 1H, 4-H),1.96 (m, 1H, cPr), 1.36 (s, 3H, 15-CH₃), 1.05 (s, 3H, 18-CH₃), 0.81 (d,3H, 17-CH₃, J=7 Hz), 0.62 (d, 3H, 16-CH₃, J=7 Hz), 0.32, 0.16 (2m, 4H,cPr). MS-ESI (m/z): 548 (MH⁺), 546 (M-H)⁻.

(b): ¹H NMR (500 MHz, DMSO-d₆, δ, ppm, inter alia): 6.13 (m, 1H, 19-H,J=11 and 18 Hz), 5.54 (d, 1H, 14-H, J=8 Hz), 5.05 (m, 1H, 20-H), 4.70(d, 1H, 2′-OH; J=5 Hz), 4.47 (d, 1H, 11-OH, J=6 Hz), 3.68 (m, 1H, 2′-H),3.45-3.15 (m, 3H, 11-H, 22-H) 2.86 (m, 1H, 4′-H), 2.71 (m, 1H, 1′-H),2.39 (bs, 1H, 4-H), 1.96 (m, 1H, cPr), 1.35 (s, 3H, 15-CH₃), 1.05 (s,3H, 18-CH₃), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.62 (d, 3H, 16-CH₃, J=7 Hz),0.32, 0.15 (2m, 4H, cPr). MS-ESI (m/z): 548 (MH⁺), 546 (M-H)⁻.

Step C.14-O-{[(1R,2R,5S*)-5-Cyclopropylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,5R*) diastereomer hydrochloride

14-O-{[(1R,2R,5S*)-4-Cyclopropylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R*)diastereomer (34 mg, 0.062 mmol) was treated according to the method ofExample 1 Step C to obtain14-O-{[(1R,2R,5R*)-4-Cyclopropylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,5S*) diastereomer hydrochloride (24 mg, 66% yield)as colorless solid.

¹H NMR (500 MHz, DMSO₆, δ, ppm, inter alia): 6.13 (m, 1H, 19-H, J=11 and18 Hz), 5.55 (d, 1H, 14-H, J=8 Hz), 5.05 (m, 1H, 20-H), 5.00 (m, 1H,2′-OH), 4.54 (d, 1H, 11-OH, J=6 Hz), 3.66 (m, 1H, 2′-H), 3.45-3.05 (m,5H, 11-H, 22-H, 1′-H, 5′-H), 2.41 (bs, 1H, 4-H), 1.36 (s, 3H, 15-CH₃),1.06 (s, 3H, 18-CH₃), 0.82 (d, 3H, 17-CH₃, J=7 Hz), 0.63 (m, 7H, 16-CH₃, cPr). MS-ESI (m/z): 548 (MH⁺), 582 (MCl^(−).)

Example23—14-O-{[(1R,2R,4S*)-4-Cyclopropylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,4R*) diastereomer hydrochloride

14-O-{[(1R,2R,4S*)-4-Cyclopropylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,4R*)diastereomer (10 mg, 0.018 mmol) from Example 21 Step B was treatedaccording to the method of Example 1 Step C to obtain the title compound(20 mg, quantitative yield, uncorrected) as colorless solid.

¹H NMR (500 MHz, DMSO-d₆, δ, ppm, inter alia): 8.6 (bs, 2H, NH₂ ⁺), 6.13(m, 1H, 19-H, J=11 and 18 Hz), 5.55 (d, 1H, 14-H, J=8 Hz), 5.26 (m, 1H,2′-OH), 5.05 (m, 1H, 20-H), 4.52 (d, 1H, 11-OH, J=6 Hz), 3.93 (m, 1H,2′-H), 3.45-3.20 (m, 4H, 11-H, 22-H, 4′-H), 2.95 (m, 1H, 1′-H), 2.64 (m,1H, cPr), 2.40 (bs, 1H, 4-H), 1.36 (s, 3H, 15-CH₃), 1.05 (s, 3H,18-CH₃), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.74 (m, 4H, cPr), 0.62 (d, 3H,16-CH₃, J=7 Hz). MS-ESI (m/z): 548 (MH⁺), 582 (MCl⁻).

Example24—14-O-{[(1R,2R,5R*)-2-Hydroxy-5-morpholin-4-yl-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,5S*) diastereomer hydrochloride Step A.14-O-{[(1R,2R,5R*)-2-Hydroxy-5-morpholin-4-yl-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5S*)diastereomer and14-O-{[(1R,2R,5S*)-2-Hydroxy-5-morpholin-4-yl-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R*)diastereomer

14-O-{[(1R,2R)-2-Hydroxy-4-oxo-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer and14-O-{[(1R,2R)-2-hydroxy-5-oxo-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer (270 mg, 0.533 mmol) from Example 22 Step A was reactedwith morpholine (0.05 ml, 0.533 mmol) in 10 ml of dichloromethaneaccording to the method of Example 19 Step A. After addition of sodiumcyanoborohydride ethanol was added (0.6 ml) and the mixture was stirredovernight at room temperature. After work up and chromatography of thereaction mixture (silica, dichloromethane/methanol=20/1)14-O-{[(1R,2R,5R*)-2-Hydroxy-5-morpholin-4-yl-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5S*)diastereomer (a) (dichloromethane/methanol=10/1, R_(f)=0.32, 23 mg, 7%yield) and14-O-{[(1R,2R,5S*)-2-Hydroxy-5-morpholin-4-yl-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R*)diastereomer (b) (dichloromethane/methanol=10/1, R_(f)0.27, 40 mg, 13%yield) were obtained.

(a): ¹H NMR (500 MHz, CDCl₃, δ, ppm, inter alia): 6.47 (m, 1H, 19-H),5.77, 5.75 (2d, 1-H, 14-H, J=8 Hz), 5.35 (dd, 1H, 20-H, J=3 and 11 Hz),5.21 (d, 1H, 20-H, J=17 Hz), 3.70 (s, 4H, morpholine), 3.40-3.15 (m, 4H,2′-H, 11-H, 22H), 2.53 (m, 5H, 1′-H, morpholine), 2.10 (bs, 1H, 4-H),1.45 (s, 3H, 15-CH₃), 1.17 (s, 3H, 18-CH₃), 0.87 (d, 3H, 17-CH₃, J=7Hz), 0.73, 0.72 (2d, 3H, 16-CH₃, J=7 Hz). MS-ESI (m/z): 578 (MH⁺), 600(MNa⁺), 576 (M-H)⁻, 612 (MCl⁻).

(b): ¹H NMR (500 MHz, CDCl₃, δ, ppm, inter alia): 6.47 (m, 1H, 19-H),5.77, 5.75 (2d, 1H, 14-H, J=8 Hz), 5.40-5.15 (m, 2H, 20-H), 3.70 (s, 4H,morpholine), 3.47 (m, 1H, 2′-H), 3.35 (m, 1H, 11-H), 3.22 (m, 2H, 22-H),2.98 (m, 1H, 1′-H), 2.54, 2.45 (2m, 4H, morpholine), 2.10 (bs, 1H, 4-H),1.45 (s, 3H, 15-CH₃), 1.17 (s, 3H, 18-CH₃), 0.88 (d, 3H, 17-CH₃, J=6Hz), 0.72 (d, 3H, 16-CH₃, J=7 Hz). MS-ESI (m/z): 578 (MH⁺), 600 (MNa⁺),576 (M-H)⁻, 612 (MCl⁻).

Step B.14-O-{[(1R,2R,5R*)-2-Hydroxy-5-morpholin-4-yl-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,5S*) diastereomer hydrochloride

14-O-{[(1R,2R,5R*)-2-Hydroxy-5-morpholin-4-yl-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5S*)diastereomer (10 mg, 0.017 mmol) was treated according to the method ofExample 1 Step C to obtain14-O-{[(1R,2R,5R*)-2-Hydroxy-5-morpholin-4-yl-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,5S*) diastereomer hydrochloride (20 mg,quantitative yield, uncorrected) as colorless solid.

¹H NMR (500 MHz, CDCl₃, δ, ppm, inter alia): 13 (bs, 1H, NH⁺), 6.47 (m,1H, 5.78, 5.76 (2d, 1H, 14-H, J=9 Hz), 5.36 (dd, 1H, 20-H, J=4 and 11Hz), 5.23 (d, 1H, 20-H; J=17 Hz), 4.40, 3.98 (2bs, 4H, morpholine),3.45-3.20 (m, 4H, 2′-H, 11-H, 22-H), 2.91, 2.56 (2m, 5H, morpholine,1′-H), 2.11 (bs, 1H, 4-H), 1.46 (s, 3H, 15-CH₃), 1.20, 1.19 (2s, 3H,18-CH₃), 0.89 (d, 3H, 17-CH₃, J=7 Hz), 0.74, 0.73 (2d, 3H, 16-CH₃, J=7Hz). MS-ESI (m/z): 578 (MH³⁰ ), 600 (MNa⁺), 612 (MCl⁻).

Example25—14-O-{[(1R,2R,5S*)-2-Hydroxy-5-morpholin-4-yl-cyclohexylsulfanyl]-acetyl}-mutilinhydrochloride+(1S,2S,5R*) diastereomer hydrochloride

14-O-{[(1R,2R,5S*)-2-Hydroxy-5-morpholin-4-yl-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R*)diastereomer (26 mg, 0.045 mmol) from Example 24 Step A was treatedaccording to the method of Example 1 Step C to obtain the title compound(15 mg, 54% yield) as colorless solid.

¹H NMR (500 MHz, CDCl₃, δ, ppm, inter alia): 12.8 (bs, 1H, NH⁺), 6.47(m, 1H, 19-H), 5.75 (m, 1H, 14-H), 5.40-5.15 (m, 2H, 20-H), 4.40, 3.98(2bs, 4H, morpholine, 2′-H), 3.50-3.15 (m, 6H, 5′-H, 11-H, 22-H,morpholine), 2.98 (m, 2H, morpholine), 2.11 (bs, 1H, 4-H), 1.46 (s, 3H,15-CH₃), 1.19 (s, 3H, 18-CH₃), 0.89 (d, 3H, 17-CH₃, J=7 Hz), 0.74 (d,3H, 16-CH₃, J=7 Hz). MS-ESI (m/z): 578 (MH⁺), 600 (MNa⁺), 612 (MCl⁻).

Example26—14-O-{[(1R,2R,5S)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilinhydrochloride+(1S,2S,5R) diastereomer hydrochloride Step A.14-O-{[(1R,2R,5S)-5-tert-Butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5R)diastereomer

To a solution of14-O-{[(1R,2R,5S)-5-tert-butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (1 g, 1.65 mmol) from Example 1 Step A in 20 ml of ethanolwas added palladium on charcoal (10%, 515 mg, 0.48 mmol) andhydrogenated overnight at room temperature. The reaction mixture wastreated with dichloromethane, filtered and the filtrate was concentratedto dryness under reduced pressure to obtain14-O-{[(1R,2R,5S)-5-tert-butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5R)diastereomer (1 g, quantitative yield) as colorless solid.

MS-ESI (m/z): 632 (MNa⁺).

Step B.14-O-{[(1R,2R,5S)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5R)diastereomer

14-O-{[(1R,2R,5S)-5-tert-butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5R)diastereomer (1 g, 1.64 mmol) was treated according to the method ofExample 1 Step B. After work up and chromatography of the reactionmixture (silica, ethyl acetate/methanol/35% ammonia solution=33/66/1)14-O-{[(1R,2R,5S)-5-amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5R)diastereomer (R_(f)=0.35, 590 mg, 71% yield) was obtained as colorlesssolid.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 5.51 (d, 1H, 14-H, J=8Hz), 4.74 (bs, 1H, 2′-OH), 4.37 (m, 1H, 11-OH), 3.49 (m, 1H, 2′-H),3.45-3.15 (m, 3H, 11-H, 22-H), 3.00 (m, 1H, 1′-H), 2.82 (m, 1H, 5′-H),2.35 (bs, 1H, 4-H), 1.34 (s, 3H, 15-CH₃), 0.85 (s, 3H, 18-CH₃), 0.80 (d,3H, 17-CH₃, J=7 Hz), 0.63 (m, 6H, 16-CH₃, 20-H).

Step C.14-O-{[(1R,2R,5S)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilinhydrochloride+(1S,2S,5R) diastereomer hydrochloride

14-O-{[(1R,2R,5S)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5R)diastereomer (590 mg, 1.16 mmol) was treated according to the method ofExample 1 Step C to obtain14-O-{[(1R,2R,5S)-5-amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilinhydrochloride+(1S,2S,5R) diastereomer hydrochloride (566 mg, 89% yield)as colorless solid.

¹H NMR (500 MHz, DMSO-d₆, δ, ppm, inter alia): 7.9 (bs, 3H, NH₃ ⁺), 5.52(d, 1H, 14-H, J=8 Hz), 3.80-3.00 (m, 6H, 2′-H, 11-H, 22-H, 1′H, 5′-H),2.35 (bs, 1H, 4-H), 1.35 (s, 3H, 15- CH₃), 0.85 (s, 3H, 18-CH₃), 0.80(d, 3H, 17-CH₃, J=7 Hz), 0.63 (m, 6H, 16-CH₃, 20-H). MS-ESI (m/z): 510(MH⁺), 544 (MCl⁻).

Example27—14-O-{[(1R,2R,5S)-5-Ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilinhydrochloride+(1S,2S,5R) diastereomer hydrochloride Step A.19,20-Dihydro-pleuromutilin thiol

A solution of 19,20-dihydro-pleuromutilin tosylate (Egger, H.;Reinshagen, H. Journal of Antibiotics 1976, 29, 915-927.) (11.5 g, 22.2mmol) in 50 ml of acetone was treated with thiourea (1.69 g, 22.2 mmol)under reflux for 1.5 hours. The reaction mixture was evaporated todryness under reduced pressure and dissolved in ethanol. The solutionwas charged with sodium metabisulfite (4.57 g, 24.0 mmol) dissolved in20 ml of water, and 100 ml of ethyl acetate. The biphasic mixture wasrefluxed for 1.5 hours under vigorous stirring. After cooling to roomtemperature the phases were separated and the aqueous phase wasextracted three times with ethyl acetate. The combined organic layerswere dried over sodium sulfate, filtered and the solvent evaporatedunder reduced pressure. After chromatography (silica, cyclohexane/ethylacetate=2/1) 19,20-dihydro-pleuromutilin thiol (cyclohexane/ethylacetate=4/3, R_(f)=0.24, 3 g, 34% yield) were obtained.

¹H NMR (500 MHz, DMSO-d₆, δ, ppm, inter alia): 5.53 (d, 1H, 14-H, J=8Hz), 4.40 (d, 1H, 11-OH, J=6 Hz), 3.36 (t, 1H, 11-H, J=6 Hz), 3.25 (m,2H, 22-H), 2.85 (t, 1H, SH, J=8 Hz), 2.38 (bs, 1H, 4-H), 1.37 (s, 3H,15-CH₃), 0.87 (s, 3H, 18-CH₃), 0.83 (d, 3H, 17-CH₃, J=7 Hz), 0.65 (m,6H, 16-CH₃, 20-H).

Step B.14-O-{[(1R,2R,5S)-5-(tert-Butoxycarbonyl-ethyl-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5R)diastereomer

N-Ethyl-N-(cis-3,4-epoxycyclohexyl)-carbamic acid tert-butyl ester (2.8g, 11.6 mmol) from Example 7 Step B was treated with19,20-dihydro-pleuromutilin thiol (4.60 g, 11.6 mmol) according to themethod of Example 1 Step A3 over the weekend at room temperature. Afterwork up and chromatography of the reaction mixture (silica,cyclohexane/ethyl acetate=1/1)14-O-{[(1R,2R,5S)-5-(tert-Butoxycarbonyl-ethyl-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5R)diastereomer (R_(f)=0.35, 1.98 g, 27% yield) was obtained.

Step C.14-O-{[(1R,2R,5S)-5-Ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5R)diastereomer

14-O-{[(1R,2R,5S)-5-(tert-Butoxycarbonyl-ethyl-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5R)diastereomer (1.98 g, 3.10 mmol) was treated according to the method ofExample 1 Step B. After work up and chromatography of the reactionmixture (silica, ethyl acetate/methanol/35% ammonia solution=100/10/1)14-O-{[(1R,2R,5S)-5-ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5R)diastereomer (ethyl acetate/methanol/35% ammonia solution=100/100/1,R_(f)=0.7, 150 mg, 9% yield) was obtained as colorless solid.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 5.52 (d, 1H, 14-H, J=7Hz), 4.72 (m, 1H, 2′-OH), 4.36 (d, 1H, 11-OH, J=6 Hz), 3.50-3.15 (m, 4H,2′-H, 11-H, 22-H), 2.97 (m, 1H, 1′-H), 2.62 (m, 1H, 5′-H), 2.47 (m, 2H,NCH₂), 2.35 (bs, 1H, 4-H), 1.35 (s, 3H, 15-CH₃), 0.98 (m, 3H, NCH₂CH₃),0.85 (s, 3H, 18-CH₃), 0.80 (d, 3H, 17-CH₃, J=7 Hz), 0.63 (m, 6H, 16-CH₃,20-H). MS-ESI (m/z): 538 (MH⁺), 560 (MNa⁺), 536 (M-H)⁻, 572 (MCl⁻).

Step D.14-O-{[(1R,2R,5S)-5-Ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilinhydrochloride+(1s, 2S,5R) diastereomer hydrochloride

14-O-{[(1R,2R,5S)-5-Ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5R)diastereomer (38 mg, 0.071 mmol) was treated according to the method ofExample 1 Step C to obtain the title compounds (40 mg, quantitativeyield) as colorless solid.

¹H NMR (400 MHz, DMSO-d₆, δ, ppm, inter alia): 5.53 (d, 1H, 14-H, J=8Hz), 4.97 (m, 1H, 2′-OH), 4.40 (d, 1H, 11-OH, J=6 Hz), 3.64 (m, 1H,2′-H), 3.45-3.20 (m, 3H, 11-H, 22-H), 2.98 (m, 1H, 1′-H), 2.94 (m, 1H,5′-H), 2.88 (m, 2H, NCH₂), 2.37 (bs, 1H, 4-H), 1.34 (s, 3H, 15-CH₃),0.98 (t, 3H, NCH₂CH₃, J=7 Hz), 0.85 (s, 3H, 18-CH₃), 0.80 (d, 3H,17-CH₃, J=7 Hz), 0.63 (m, 6H, 16-CH₃, 20-H). MS-ESI (m/z): 538 (MH⁺).

Example28—14-O-{[(1R,2R,5R)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5S)diastereomer Step A.14-O-{[(1R,2R,5S)-5-(tert-Butyl-dimethyl-silyloxy)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5R)diastereomer

tert-Butyl-dimethyl-(cis-3,4-epoxycyclohexyloxy)-silane (864 mg, 3.78mmol) from Example 4 Step A was treated with 19,20-dihydro-pleuromutilinthiol (1.5 g, 3.78 mmol) from Example 27 Step A according to the methodof Example 1 Step A3. After work up and chromatography of the reactionmixture (silica, cyclohexane/ethyl acetate=3:1)14-O-{[(1R,2R,5S)-5-(tert-butyl-dimethyl-silyloxy)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5R)diastereomer (cyclohexane/ethyl acetate 1/1, R_(f)=0.45, 1.2 g, 51%yield) was obtained as colorless solid.

¹H NMR (500 MHz, DMSO-d₆, δ, ppm, inter alia): 5.50 (d, 1H, 14-H, J=8Hz), 4.81 (m, 1H, 2′-OH), 4.38 (d, 1H, 11-OH, J=6 Hz), 3.88 (m, 1H,5′-H), 3.50-3.20 (m, 4H, 2′-H, 11-H, 22-H), 2.95 (m, 1H, 1′-H), 2.34(bs, 1H, 4-H), 1.34 (s, 3H, 15-CH₃), 0.84 (m, 12H, 18-CH₃, tert-butyl),0.80 (d, 3H, 17-CH₃, J=7 Hz), 0.63 (m, 6H, 16-CH₃, 20-H), 0.02 (s, 6H,Si(CH₃)₂).

Step B.14-O-{[(1R,2R,5S)-2,5-Dihydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5R)diastereomer

14-O-{[(1R,2R,5S)-5-(tert-butyl-dimethyl-silyloxy)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5R)diastereomer (1.2 g, 1.92 mmol) was treated according to the method ofExample 4 Step C. After work up and chromatography of the reactionmixture (silica, cyclohexane/ethyl acetate=1/4)14-O-{[(1R,2R,5S)-2,5-dihydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5R)diastereomer (cyclohexane/ethyl acetate=1:1, R_(f)=0.2, 720 mg, 73%yield) was attained as colorless solid.

¹H NMR (500 MHz, DMSO-d₆, δ, ppm, inter alia): 5.50 (d, 1H, 14-H, J=8Hz), 4.74 (d, 1H, 2′-OH, J=3 Hz), 4.42 (m, 1H, 5′-OH), 4.38 (d, 1H,11-OH, J=6 Hz), 3.67 (m, 1H, 5′-H), 3.50-3.20 (m, 4H, 2′-H, 11-H, 22-H),2.96 (m, 1H, 1′-H), 2.34 (bs, 1H, 4-H), 1.34 (s, 3H, 15-CH₃), 0.84 (s,3H, 18-CH₃), 0.80 (d, 3H, 17-CH₃, J=7 Hz), 0.63 (m, 6H, 16-CH₃, 20-H).

Step C.14-O-{[(1R,2R,5S)-2-Hydroxy-5-methanesulfonyloxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5R)diastereomer

14-O-{[(1R,2R,5S)-2,5-dihydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5R)diastereomer (720 mg, 1.41 mmol) was treated according to the method ofExample 4 Step D. After work up and chromatography of the reactionmixture (silica, cyclohexane/ethyl acetate=1/1)14-O-{[(1R,2R,5S)-2-hydroxy-5-methanesulfonyloxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5R)diastereomer (cyclohexane/ethyl acetate=1/2, R_(f)=0.4, 640 g, 77%yield) was obtained as colorless solid.

¹H NMR (500 MHz, DMSO-d₆, δ, ppm, inter alia): 5.51 (d, 1H, 14-H, J=8Hz), 4.79 (m, 1H, 5′-H), 4.38 (bs, 1H, 11-OH), 3.60-3.20 (m, 7H, 2′-H,11-H, 22-H, SO₂CH₃), 2.93 (m, 1H, 1′-H), 2.35 (bs, 1H, 4-H), 1.35 (s,3H, 15-CH₃), 0.84 (s, 3H, 18-CH₃), 0.80 (d, 3H, 17-CH₃, J=7 Hz), 0.63(m, 6H, 16-CH₃, 20-H).

Step D.14-O-{[(1R,2R,5R)-5-Azido-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5S)diastereomer

14-O-{[(1R,2R,5S)-2-hydroxy-5-methanesulfonyloxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5R)diastereomer (640 mg, 1.09 mmol) was treated with sodium azide accordingto the method of Example 4 Step E. After work up crude14-O-{[(1R,2R,5R)-5-azido-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5S)diastereomer (quantitative yield, cyclohexane/ethyl acetate=1/2,R_(f)=0.7) was obtained which was directly used for the next step.

Step E.14-O-{[(1R,2R,5R)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5S)diastereomer

14-O-{[(1R,2R,5R)-5-Azido-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5S)diastereomer (584 mg, 1.09 mmol) was treated with triphenylphosphinc(342 mg, 1.30 mmol) according to the method of Example 4 Step F. Afterwork up and chromatography (silica,dichloromethane/methanol/i-propanol/water/acetic acid=80/20/6/3/2) withsubsequent basic extraction14-O-{[(1R,2R,5R)-5-amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilin+(1S,2S,5S)diastereomer (silica, dichloromethane/methanol/35% ammoniasolution=100/10/1, R_(f)=0.3, 50.5 mg, 9% yield) was obtained ascolorless foam.

¹H NMR (500 MHz, DMSO-d₆, δ, ppm, inter alia): 5.51 (d, 1H, 14-H, J=8Hz), 4.77 (m, 1H, 2′-OH), 4.38 (d, 1H, 11-OH, J=8 Hz), 3.60-3.15 (m, 4H,2′-H, 11-H, 22-H), 2.60 (m, 1H, 1′-H), 2.50 (m, 1H, 5′-H), 2.35 (bs, 1H,4-H), 1.34 (s, 3H, 15-CH₃), 0.84 (s, 3H, 18-CH₃), 0.80 (d, 3H, 17-CH₃,J=7 Hz), 0.63 (m, 6H, 16-CH₃, 20-H). MS-ESI (m/z): 510 (MH⁺).

Example29—14-O-{[(1R,2R)-4-Aminomethyl-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilindiasteromers+(1S,2S) diastereomers Step A.Cyclohex-3-enylmethyl-carbamic acid tert-butyl ester

To a solution of C-Cyclohex-3-enyl-methylamine (3.28 g, 29.5 mmol) andN-methyl-morpholine (2.98 g, 29.5 mmol) in 70 ml of anhydrousdichloromethane was added di-tert-butyldicarbonate (6.44 g, 29.5 mmol)under cooling. The resulting mixture was stirred for 20 hours at roomtemperature and the solvent was removed under reduced pressure. Theresidue was diluted with ethyl acetate and washed with 1N HCl. Theaqueous phase was extracted three times with ethyl acetate and thecombined organic phases were washed with water and brine. The resultingorganic phase was dried over magnesium sulfate, filtered and the solventwas removed under reduced pressure to yield 6.57 g of a brown oil.Column chromatography (silica, petrol ether/ethyl acetate=12/1 to 8/1)resulted in cyclohex-3-enylmethyl-carbamic acid tert-butyl ester (petrolether/ethyl acetate=10/1, R_(f)=0.62, 3.06 g, 49% yield) as colorlesssolid.

¹H NMR (200 MHz, DMSO-d₆, δ, ppm, inter alia): 6.90-6.83 (m, 1H, NH),5.66-5.63 (m, 2H, olef. H), 2.86 (t, 2H, CH₂N, J=6 Hz), 2.21-1.50 (m,6H, 2×CH₂), 2.21-0.95 (m, 16H, tert-butyl, 3×CH₂ and CH).

Step B. (7-Oxa-bicyclo[4.1.0]hept-3-yl)-carbamic acid tert-butyl ester

To a solution of cyclohex-3-enylmethyl-carbamic acid tert-butyl ester(1.5 g, 7.10 mmol) in 20 ml of anhydrous dichloromethane was added3-chloroperoxybenzoic acid (2.45 g, 14.2 mmol) under cooling. Theresulting mixture was stirred for 19 hours at room temperature andwashed with saturated sodium bicarbonate and 0.5M aqueous solution ofsodium thiosulphate. The aqueous phase was extracted three times withdichloromethane and the combined organic phases were washed with brine.The resulting organic phase was dried over magnesium sulfate, filteredand the solvent was removed under reduced pressure to yield 1.48 g ofcrude product. Column chromatography of 3.17 g of crude product (silica,petrol ether/ethyl acetate=3/1) resulted in(7-oxa-bicyclo[4.1.0]hept-3-ylmethyl)-carbamic acid tert-butyl ester(R_(f)=0.19, 2.68 g, 81% yield) as colorless solid.

¹H NMR (200 MHz, DMSO-d₆, δ, ppm, inter alia): 6.84-6.78 (m, 1H, NH),3.09-3.03 (m, 2H, CHO), 2.71-2.70 (m, 2H, CH₂N), 1.37 (s, 9H,tert-butyl). MS-ESI (m/z): 250 (MNa⁺), 477 (2MNa⁺).

Step C.14-O-{[(1R,2R)-4-(tert-Butoxycarbonylamino-methyl)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilindiasteromers+(1S,2S) diastereomers

To a solution of (7-oxa-bicyclo[4.1.0]hept-3-ylmethyl)-carbamic acidtert-butyl ester (1.34 g, 5.90 mmol) and Pleuromutilin thiol (2.32 g,5.90 mmol) in 25 ml of methanol was added 2M NaOH (2.95 ml, 5.90 mmol)drop wise under cooling. The resulting mixture was stirred at roomtemperature overnight and the solvent was removed under reducedpressure. The residue was diluted with ethyl acetate and washed withbrine. The aqueous phase was extracted three times with ethyl acetateand the combined organic phases were washed with brine. The resultingorganic phase was dried over magnesium sulfate, filtered and the solventwas removed under reduced pressure to yield 3.86 g of a crude product.Column chromatography (silica, petrol ether/ethyl acetate=1/1) resultedin14-O-{[(1R,2R)-4-(tert-butoxycarbonylamino-methyl)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilindiasteromers+(1S,2S) diastereomers (R_(f)=0.24, 2.11 g, 58% yield) ascolorless solid.

¹H NMR (500 MHz, CDCl₃, δ, ppm, inter alia): 6.32-6.40 (m, 1H, 19-H),5.80-5.71 (m, 1H, 14-H), 5.40-5.15 (m, 2H, 20-H), 4.64, 4.55 (2bs, 1H,NH), 3.79-3.67, 3.58-3.45 (2m, 1H, 2′-H), 3.40-3.31 (m, 1H, 11-H),3.29-3.11 (m, 2H, 22-H), 3.10-2.92 (m, 2H, CH₂N), 2.89-2.77, 2.74-2.64(2m, 1H, 1′-H), 1.45 (s, 3H, 15-CH₃), 1.43 (s, 9H, tert-butyl), 1.17 (s,3H, 18-CH₃), 0.78-0.66 (m, 3H, 16-CH₃). MS-ESI (m/z): 644 (MNa³⁰ ).

Step D.14-O-{[(1R,2R)-4-Aminomethyl-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilindiasteromers+(1S,2S) diastereomers

To a solution of14-O-{[(1R,2R)-4-(tert-butoxycarbonylamino-methyl)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilindiasteromers+(1S,2S) diastereomers (1.14 g, 1.83 mmol) in 20 ml ofanhydrous dichloromethane was added 20 ml of 1M HCl in diethyl etherdrop wise under cooling. The resulting mixture was stirred at roomtemperature for two days and the solvent was removed under reducedpressure. The residue was diluted with dichloromethane and washed with asaturated solution of sodium bicarbonate. The aqueous phase wasextracted three times with dichloromethane. The resulting organic phaseswere combined and dried over magnesium sulfate, filtered and the solventwas removed under reduced pressure. Column chromatography (silica,dichloromethane/methanol=5/1 to 1/1) resulted in14-O-{[(1R,2R)-4-aminomethyl-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilindiasteromers+(1S,2S) diastereomers (39 mg, 8% yield) as colorless solid.

¹H NMR (400 MHz, CDCl₃, δ, ppm, inter alia): 6.58-6.38 (m, 1H, 19-H),5.87-5.68 (m, 1H, 14-H), 5.44-5.12 (m, 2H, 20-H), 4.52-4.34 (m, 1H,1′-H), 2.68-2.52 (m, 2H, CH₂N), 1.46 (s, 3H, 15-CH₃), 1.19 (s, 3H,18-CH₃), 0.89 (d, 3H, 17-CH₃, J=7 Hz), 0.74 (d, 3H, 16- CH₃, J=7 Hz).MS-ESI (m/z): 544 (MNa⁺).

Example 30—14-O-{[5-Amino-2-chloro-cyclohexylsulfanyl]-acetyl}-mutilinacetate and 14-O-{[4-Amino-2-chloro-cyclohexylsulfanyl]-acetyl}-mutilinacetate Step A. (1R,3S,6S)-(7-thia-bicyclo[4.1.0]hept-3-yl)-carbamicacid tert-butyl ester+(1S,3R,6R) diastereomer

To a solution of syn-3,4-epoxycyclohexyl-carbamic acid tert-butyl ester(3.55 g, 16.6 mmol) and tetrabutylammonium chloride (500 mg, 1.80 mmol)in 50 ml of tert-butyl methyl ether was added a solution of potassiumthiocyanate (8.07 g, 83.0 mmol) in 50 ml of water. The resulting mixturewas stirred for 7 days at room temperature and the phases wereseparated. The aqueous phase was extracted twice with ethyl acetate andthe combined organic phases were washed with brine. The resultingorganic phase was dried over magnesium sulfate, filtered and the solventwas removed under reduced pressure to yield 4.33 g of a crude product asa colorless solid. Column chromatography (silica, petrol ether/ethylacetate=7/1 to 3/1) resulted in(1R,3S,6S)-(7-thia-bicyclo[4.1.0]hept-3-yl)-carbamic acid tert-butylester+(1S,3R,6R) diastereomer (petrol ether/ethyl acetate=5/1,R_(f)=0.54, 1.88 g, 49% yield based on recovered starting material) ascolorless crystals. Fp=105-108° C.

¹H NMR (500 MHz, CDCl₃, δ, ppm, inter alia): 4.40 (bs, 1H, NH), 3.78(bs, 1H, 1-H), 3.28-3.20 (m, 1H, 3-H), 3.18-3.09 (m, 1H, 4-H), 2.54 (dd,1H, 2a-H, J=5 Hz and J=15 Hz), 2.40-2.18 (m, 2H, 5-H), 1.98-1.87 (m, 1H,2b-H), 1.86-1.72 (m, 1H, 6a-H), 1.58 (s, 9H, tert-butyl), 1.34-1.20 (m,1H, 6b-H). MS-ESI (m/z): 252 (MNa⁺).

Step B.14-O-{[(1R,5R,8R)-3-oxo-2-oxa-4-aza-bicyclo[3.3.1]non-8-ylsulfanyl]-acetyl}-mutilin+(1S,5S,8S)diastereomer

To a solution of (1R,3S,6S)-(7-thia-bicyclo[4.1.0]hept-3-yl)-carbamicacid tert-butyl ester+(1S,3R,6R) diastereomer (2.95 g, 12.9 mmol) in 160ml of dichloromethane was added p-toluene sulfonic acid (1.21 g, 6.50mmol) under cooling. The resulting mixture was stirred at roomtemperature over night and the solvent was removed under reducedpressure to yield 3.26 g of a colorless solid. The crude product wassubsequently dissolved in 150 ml of anhydrous tetrahydrofuran, andpleuromutilin tosylate (13.1 g, 24.6 mmol) followed by1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 4.2 ml, 28.2 mmol) was addedunder cooling. The resulting mixture was stirred at room temperatureovernight and water was added. The mixture was extracted four times withethyl acetate and the combined organic phases were washed with water andbrine, dried over magnesium sulfate and the solvent was removed underreduced pressure to yield 9.32 g of a colorless solid. Chromatography(silica, dichloromethane/methanol=19/1) resulted in14-O-{[(1R,5R,8R)-3-oxo-2-oxa-4-aza-bicyclo[3.3.1]non-8-ylsulfanyl]-acetyl}-mutilin+(1S,5S,8S)diastereomer (dichloromethane/methanol=20/1, R_(f)=0.45, 4.36 g, 63%yield) as colorless solid.

¹H NMR (500 MHz, CDCl₃, δ, ppm, inter alia): 6.46 (dd, 1H, 19-H, J=7 Hzand J=11 Hz), 5.77 (d, 1H, 14-H, J=8 Hz), 5.52 (bs, 1H, NH), 5.40-5.17(m, 2H, 20-H), 4.68-4.55 (m, 1H, 2′-H), 3.63 (bs, 1H, 4′-H), 3.41-3.30(m, 2H, 1′-H, 11-H), 3.27-3.12 (m, 2H, 22-H), 1.45 (s, 3H, 15-CH₃), 1.18(s, 3H, 18-CH₃), 0.89 (d, 3H, 17-CH₃, J=7 Hz), 0.73 (dd, 3H, 16- CH₃,J=2 Hz and J=7 Hz). MS-ESI (m/z): 556 (MNa⁺).

Step C. 14-O-{[5-Amino-2-chloro-cyclohexylsulfanyl]-acetyl}-mutilinacetate and 14-O-{[4-Amino-2-chloro-cyclohexylsulfanyl]-acetyl}-mutilinacetate

To a solution of14-O-{[(1R,5R,8R)-3-oxo-2-oxa-4-aza-bicyclo[3.3.1]non-8-ylsulfanyl]-acetyl}-mutilin+(1S,5S,8S)diastereomer (500 mg, 0.94 mmol) in 2.5 ml of dioxane was added 6M HCl(7 ml) under cooling. The resulting mixture was stirred for 23 hours andadded to a saturated solution of sodium bicarbonate. The resultingsolution was extracted twice with ethyl acetate and the combined organicphases were washed with brine. The resulting organic phase was driedover magnesium sulfate, filtered and the solvent was removed underreduced pressure to yield 473 mg of crude product. Column chromatography(silica, dichloromethane/methanol=10/1 containing 1% of acetic acid anddichloromethane/methanol/diisopropylether/water/acetic acid=80/20/6/3/2)resulted in 14-O-{[5-Amino-2-chloro-cyclohexylsulfanyl]acetyl}-mutilinacetate (a) (dichloromethane/methanol/diisopropylether/water/aceticacid=80/20/6/3/2, R_(f)=0.5, 178 mg, 36% yield) and14-O-{[4-Amino-2-chloro-cyclohexylsulfanyl]acetyl}-mutilin acetate (b)(dichloromethane/methanol/diisopropylether/water/aceticacid=80/20/6/3/2, R_(f)=0.43, 91 mg, 18% yield) as a colorless solids.

(a): ¹H NMR (500 MHz, CDCl₃, δ, ppm, inter alia): 6.46 (dd, 1H, 19-H,J=11 Hz and J=17 Hz), 5.78 (d, 1H, 14-H, J=8 Hz), 5.45-5.15 (m, 2H,20-H), 4.32 (m, 1H, 2′-H), 3.41 -3.30 (m, 2H, 11-H, 1′-H), 3.28-3.14 (m,2H, 22-H), 3.13-3.00 (m, 1H, 5′-H), 2.02 (s, 3H, CH₃ of acetate), 1.46(s, 3H, 15-CH₃), 1.19 (s, 3H, 18-CH₃), 0.89 (d, 3H, 17-CH₃, J=7 Hz),0.73 (d, 3H, 16-CH₃, J=7 Hz). MS-ESI (m/z): 526 (MH⁺).

(b): ¹H NMR (500 MHz, CDCl₃, δ, ppm, inter alia): 6.49 (dd, 1H, 19-H,J=11 Hz and J=17 Hz), 5.77 (d, 1H, 14-H, J=8 Hz), 5.40-5.15 (m, 2H,20-H), 3.88-3.73 (m, 1H, 2′-H), 3.50-3.15 (m, 3H, 11-H, 22-H), 3.00-2.70(m, 2H, 1′-H, 4′-H), 2.03 (s, 3H, CH₃ of acetate), 1.46 (s, 3H, 15-CH₃),1.18 (s, 3H, 18-CH₃), 0.88 (d, 3H, 17-CH₃, J=7 Hz), 0.74 (d, 3H, 16-CH₃,J=7 Hz). MS-ESI (m/z): 526 (MH⁺).

Example31—14-O-[(4-Amino-1-hydroxy-cyclohexylmethylsulfanyl)-acetyl]-mutilinhydrochloride Step A. (1-Oxa-spiro[2.5]oct-6-yl)-carbamic acidtert-butyl ester

(4-Methylene-cyclohexyl)-carbamic acid tert-butyl ester (Raju, B. et al,Bioorganic and Medicinal Chemistry Letters 2004, 14(12), 3103-3107) (2.3g, 10.9 mmol) was treated with 3-chloroperbenzoic acid (70% purity, 3.76g, 21.8 mmol, uncorrected) according to the method of Example 7 Step B.After work up the title compound was obtained (2.3 g, 93% yield) as ayellow solid.

¹H NMR (DMSO-d₆, 400 MHz, δ, ppm): 6.77 (bd, 1H, NH, J=7 Hz), 3.35 (m,1H), 2.55, 2.51 (2s, 2H), 1.86-1.75 (m, 2H), 1.73-1.67 (m, 2H),1.47-1.28 (m, 2H), 1.36 (s, 9H, tert-butyl), 1.22-1.13 (m, 2H).

Step B.14-O-[(4-tert-Butoxycarbonylamino-1-hydroxy-cyclohexylmethylsulfanyl)-acetyl]-mutilin

Pleuromutilin thiol (2.6 g, 6.59 mmol) was treated with(1-Oxa-spiro[2.5]oct-6-yl)-carbamic acid tert-butyl ester (1 g, 4.40mmol) according to the method of Example 1 Step A3. After workup andchromatography of the mixture (silica, toluene/ethyl acetate=5/1−>3/1)the title compound (toluene/ethyl acetate=1/1, R_(f)=0.44, 0.41 g, 15%yield, uncorrected) was obtained as a colorless foam.

¹H NMR (DMSO-d₆, 500 MHz, δ, ppm, inter alia): 6.66 (d, 1H, NH, J=8 Hz),6.14 (dd, 1H, 19-H, J=18 Hz and 11 Hz), 5.54 (d, 1H, 14-H, J=8 Hz),5.10-5.02 (m, 2H, 20-H), 4.49 (bs, 1H, 11-OH), 4.23 (bs, 1H), 3.46-3.28(m, 3H), 3.08 (bs, 3H, 4′-H), 2.60 (s, 2H, COHCH₂S), 2.40 (bs, 1H, 4-H),2.18 (m, 1H), 2.12-2.02 (m, 3H), 1.69-1.54 (m, 3H), 1.52-1.43 (m, 3H),1.35 (s, 3H, 15-CH₃), 1.35 (s, 9H, tert-butyl), 1.32-1.21 (m, 4H), 1.08(s, 3H, 18-CH₃), 1.14-0.98 (m, 1H), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.62(d, 3H, 16-CH₃, J=7 Hz). MS-ESI (m/z): 644 (MNa⁺).

Step C.14-O-[(4-Amino-1-hydroxy-cyclohexylmethylsulfanyl)-acetyl]-mutilin

14-O-[(4-tert-Butoxycarbonylamino-1-hydroxy-cyclohexylmethylsulfanyl)-acetyl]-mutilin(0.36 g, 0.58 mmol) was treated with trifluoroacetic acid (0.72 ml)according to the method of Example 1 Step B. After workup andchromatography of the mixture (silica, ethyl acetate/methanol/NH₄OH(25%)=50/50/1) the title compound (R_(f)=0.04, 0.13 g, 43% yield) wasobtained as colorless foam.

¹H NMR (DMSO-d₆, 400 MHz, δ, ppm, inter alia): 6.13 (dd, 1H, 19-H, J=18Hz and 11 Hz), 5.53 (d, 1H, 14-H, J=8 Hz), 5.10 (d, 1H, 20-H, J=2 Hz),5.02 (d, 1H, 20-H, J=2 Hz), 4.49 (bs, 1H), 4.21 (bs, 1H), 3.42 (m, 1H),3.23 (q, 2H, H-22, J=14 Hz), 2.60 (s, 2H, COHCH₂S), 2.47-2.36 (m, 2H),2.23-2.14 (m, 1H), 2.12-2.04 (m, 3H), 1.68-1.54 (m, 3H), 1.41-1.20 (m,5H), 1.35 (s, 3H, 15-CH₃), 1.05 (s, 3H, 18-CH₃), 1.03 (m, 1H), 0.81 (d,3H, 17-CH₃, J=7 Hz), 0.62 (d, 3H, 16-CH₃, J=7 Hz). MS-ESI (m/z)=522(MH⁺), 544 (MNa⁺).

Step D.14-O-[(4-Amino-1-hydroxy-cyclohexylmethylsulfanyl)-acetyl]-mutilinhydrochloride

A solution of14-O-[(4-amino-1-hydroxy-cyclohexylmethylsulfanyl)-acetyl]-mutilin (0.1g, 0.19 mmol) in 1 ml dioxane was treated with aqueous hydrochloric acid(0.05 M, 11.6 ml, 0.58 mmol) under stirring according to the method ofExample 1 Step C. After 1 hour the mixture was lyophilized overnight togive the title compound (107 mg, 99% yield) as white solid.

¹H NMR (DMSO-d₆, 400 MHz, δ, ppm, inter alia): 7.88 (s, 3H, NH₃ ⁺), 6.13(dd, 1H, 19-H, J=18 Hz and 11 Hz), 5.52 (d, 1H, J=8 Hz), 5.08 (dd, 1H,20-H, J=5 Hz and 18 Hz), 5.03 (dd, 1H, 20-H, J=5 Hz and 11 Hz), 3.25 (q,2H, 22-H, J=14 Hz), 2.85 (m, 1H, 4′-H), 2.62 (s, 2H, COHCH₂S), 2.40 (s,1H, 4-H), 2.23-2.03 (m, 4H), 1.71-1.56 (m, 6H), 1.47 (m, 1 H), 1.36 (s,3H, 15-CH₃), 1.41-1.19 (m, 4H), 1.05 (s, 3H, 18-CH₃), 1.00 (m, 1H), 0.81(d, 3H, 17-CH₃, J=7 Hz), 0.62 (d, 3H, 16-CH₃, J=7 Hz).

Example32—14-O-{[(1R,2R)-2-Hydroxy-5-(3-methylamino-propyl)-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer and14-O-{[(1R,2R)-2-Hydroxy-4-(3-methylamino-propyl)-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer Step A. 3-Cyclohex-3-enyl-N-methyl-propionamide

Methyl amine (8 M in EtOH, 75 ml, 600 mmol) was added to a mixture of3-cyclohex-3-enyl-propionic acid methyl ester (German patent DE 4023848A1 19920130) (20.0 g, 119 mmol) in 75 mL of methanol. The mixture wasstirred at room temperature for one day. Additional Methyl amine (8 M inEtOH, 40 ml, 320 mmol) was added to the mixture and stirring continuedfor one more day. The mixture was concentrated and the residue was takenup in ethyl acetate, washed with 0.5 M aqueous HCl, dried and strippedof the solvent to give the title compound (19.50 g, 98% yield,uncorrected) as a pale orange solid.

¹H NMR (CDCl₃, 200 MHz, δ, ppm): 5.58 (s, 2H, olefinic H), 5.57 (m, 1H,NH), 2.74 (d, 3H, NCH₃, J=5 Hz), 2.20-1.90 (m, 5H), 1.75-1.40 (m, 5H),1.15 (m, 1H).

Step B. (3-Cyclohex-3-enyl-propyl)-methyl-amine

A solution of 3-cyclohex-3-enyl-N-methyl-propionamide (15.5 g, 92.7mmol) in 55 ml of tetrahydrofuran was added dropwise over a period of 25min to a suspension of lithium aluminium hydride (95% purity, 5.3 g, 139mmol, corrected) in 120 ml of tetrahydrofuran at 0° C. under stirring.The mixture was refluxed for 4 hours, stirred overnight at roomtemperature and quenched with 2 M aqueous NaOH, diluted withtetrahydrofuran, stirred and filtered. The filtrate was concentrated andthe residue was acidified with 1 M aqueous HCl and washed withdichloromethane. The aqueous phase was basified with 1 M aqueous NaOH,and extracted with ethyl acetate. The organic extract was dried andconcentrated to obtain the title compound (9.38 g, 66% yield) as paleyellow oil.

¹H NMR (CDCl₃, 200 MHz, δ, ppm): 5.58 (d, 2H, olefinic H, J=2 Hz), 2.50(t, 2H, J=7 Hz), 2.36 (s, 3H, NCH₃), 2.15-1.90 (m, 3H), 1.75-1.05 (m,9H).

Step C. (3-Cyclohex-3-enyl-propyl)-methyl-carbamic acid tert-butyl ester

Ethyl-diisopropyl-amine (11.3 ml, 66.0 mmol) anddi-tert-butyl-dicarbonate (14.4 g, 66.0 mmol) were added to a solutionof (3-cyclohex-3-enyl-propyl)-methyl-amine (7.50 g, 48.9 mmol) in 75 mlof dioxane. The mixture was stirred at room temperature for 3 days,diluted with ethyl acetate and washed with cold 0.1 M aqueous HCl, andsaturated aqueous sodium bicarbonate, dried over sodium sulfate andconcentrated in vacuo to give a mixture. After chromatography of themixture (silica, dichloromethane) the title compound (14.24 g,quantitative yield, uncorrected) was obtained as pale yellow oil.

¹H NMR (CDCl₃, 200 MHz, δ, ppm): 5.65 (d, 2H, olefinic H, J=2 Hz), 3.19(t, 2H, CH₂N, J=7 Hz), 2.84 (s, 3H, NCH₃), 2.15-1.90 (m, 3H), 1.75-1.30(m, 5H), 1.45 (s, 9H, tert-butyl), 1.30-1.05 (m, 3H).

Step D. Methyl-[3-(7-oxa-bicyclo[4.1.0]hept-3-yl)-propyl]-carbamic acidtert-butyl ester

(3-Cyclohex-3-enyl-propyl)-methyl-carbamic acid tert-butyl ester (14.24g, 56.2 mmol) was treated with 3-chloroperbenzoic acid (70% purity, 14.8g, 60 mmol, corrected) according to the method of Example 7 Step B andstirred for 3 hours at room temperature. After work up the titlecompound (14.6 g, 96% yield, uncorrected) was obtained as pale yellowoil.

¹H NMR (CDCl₃, 200 MHz, δ, ppm): 3.15-3.00 (m, 4H), 2.75 (s, 3H, NCH₃),2.15-0.70 (m, 11H), 1.38 (s, 9H, tert-butyl).

Step E.14-O-{{(1R,2R)-5-[3-(tert-Butoxycarbonyl-methyl-amino)-propyl]-2-hydroxy-cyclohexylsulfanyl}-acetyl}-mutilin+(1S,2S)diastereomer and14-O-{{(1R,2R)-4-[3-(tert-Butoxycarbonyl-methyl-amino)-propyl]-2-hydroxy-cyclohexylsulfanyl}-acetyl}-mutilin+(1S,2S)diastereomer

Methyl-[3-(7-oxa-bicyclo[4.1.0]hept-3-yl)-propyl]-carbamic acidtert-butyl ester (3.00 g, 11.1 mmol) was treated with pleuromutilinthiol (6.57 g, 16.7 mmol) according to the method of Example 1 Step A3and stirred for 3 days at room temperature. After workup andchromatography of the mixture (silica, cyclohexane/ethyl acetate=1:1) amixture of the title compounds (R_(f)=0.29, 3.20 g, 43% yield,uncorrected) was obtained as white foam. The mixture was taken to thenext step.

Step F.14-O-{[(1R,2R)-2-Hydroxy-5-(3-methylamino-propyl)-cyclohexylsufanyl]-acetyl}-mutilin+(1S,2S)diastereomer and14-O-{[(1R,2R)-2-Hydroxy-4-(3-methylamino-propyl)-cyclohexysulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer

HCl (4 M in dioxane, 5.30 ml, 21.2 mmol) was added to the mixture ofcompounds from Example 32 Step E (5.63 g, 8.48 mmol, uncorrected) in 50ml of dioxane. The mixture was stirred for 4 hours and stripped of thesolvent. The residue was portioned between dichloromethane and saturatedaqueous bicarbonate and the organic layer was separated, dried andstripped of the solvent to give a mixture. After chromatography (silica,dichloromethane/methanol/28%-aq. NH₃=91/6/3)14-O-{[(1R,2R)-2-Hydroxy-5-(3-methylamino-propyl)-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer (a) (dichloromethane/methanol/28%-aq. NH₃=88/8/4,R_(f)=0.18, 117 mg, 3% yield, uncorrected) and14-O-{[(1R,2R)-2-Hydroxy-4-(3-methylamino-propyl)-cyclohexylsulfanyl]-acetyl}-mutilin(b) (dichloromethane/methanol/28%-aq. NH₃=88/8/4, R_(f)=0.10, 172 mg, 4%yield, uncorrected) were obtained as white foams.

(a): ¹H NMR: (400 MHz, DMSO-d₆, δ, ppm, inter alia): 6.12 (dd, 1H, 19-H,J=11 Hz and 18 Hz), 5.53 (d, 1H, 14-H, J=8 Hz), 5.1-5.0 (m, 2H, 20-H),4.74 (bs, 1H, 2′-OH), 4.49 (bs, 1H, 11-OH), 3.59 (m, 1H), 3.42 (m, 1H,11-H), 3.20 (m), 2.90 (m, 1H), 2.42-2.36 (m, 3H), 2.24 (s, 3H, CH₃-N),2.21-2.01 (m), 1.72-1.56 (m), 1.55-1.10 (m), 1.36 (s, 3H, 15-CH₃), 1.05(s, 3H, 18-CH₃), 1.00 (m, 1H), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.62 (d, 3H, 16-CH₃, J=7 Hz).

(b): ¹H NMR: (500 MHz, DMSO-d₆, δ, ppm, inter alia): 6.13 (m, 1H, 19-H),5.54 (d, 1H, H-14, J=9 Hz), 5.10-5.00 (m, 2H, 20-H), 4.74 (bs, 1H,2′-OH), 4.48 (bs, 1H, 11-OH), 3.70 (bs, 1H, 2′-H), 3.41 (m, 1H, 11-H),3.20 (m, 2H, H-22), 2.85 (m, 1H), 2.43-2.37 (m, 3H), 2.24 (s, 3H,CH₃-N), 2.22-1.89 (m), 1.70-0.96 (m), 1.35 (s, 3H, 15-CH₃), 1.05 (s, 3H,18-CH₃), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.62 (d, 3H, 16-CH₃, J=7 Hz).

Example33—14-O-{[(1R,2R)-5-(3-Amino-propyl)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer and14-O-{[(1R,2R)-4-(3-Amino-propyl)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer Step A.N-tert-Butoxycarbonyl-(3-cyclohex-3-enyl-propyl)-carbamic acidtert-butyl ester

Sodium hydride (60% in mineral oil, 2.5 g, 62.5 mmol, corrected) wasadded in several portions over a period of 15 min to an ice-cold mixtureof di-tert-butyl-iminodicarboxylate (22.0 g, 100 mmol) in 60 ml ofdimethyl formamide and 180 ml of tetrahydrofuran under stirring. Asolution of toluene-4-sulfonic acid 3-cyclohex-3-enyl-propyl ester(Marvell, E.; Sturmer, D.; Kunston, R. Journal of Organic Chemistry1968, 33, 2991-2993) (14.8 g, 50.0 mmol) in a mixture of 15 ml ofdimethyl formamide and 45 ml of tetrahydrofuran was charged to itdropwise over 30 minutes. The mixture was stirred for 7 hours at 70° C.and 16 hours at room temperature, diluted with water and extracted withtert-butyl methyl ether. The organic extract was washed with water,brine and stripped of the solvent to give a mixture. Afterchromatography of the mixture (silica, toluene/cyclohexane=75/25) thetitle compound (ethyl acetate/toluene=15/85, R_(f)=0.19, 14.7 g, 86%yield, uncorrected) was obtained as clear yellow oil.

¹H NMR (CDCl₃, 200 MHz, δ, ppm): 5.57 (d, 2H, olefinic H, J=2 Hz), 3.46(t, 2H, CH₂N, J=8 Hz), 2.15-1.90 (m, 3H), 1.75-1.30 (m, 5H), 1.43 (s,18H, tert-butyl), 1.30-1.00 (m, 3H).

Step B.N-tert-Butoxycarbonyl-[3-(7-oxa-bicyclo[4.1.0]hept-3-yl)-propyl]-carbamicacid tert-butyl ester

N-tert-Butoxycarbonyl-(3-cyclohex-3-enyl-propyl)-carbamic acidtert-butyl ester (12.2 g, 29.4 mmol, corrected) was treated with3-chloroperbenzoic acid (70% purity, 8.7 g, 35.3 mmol, corrected)according to the method of Example 7 Step B and stirred at roomtemperature for 4.5 hours. After work up the title compound (9.38 g, 90%yield) was obtained as light yellow oil.

¹H NMR (CDCl₃, 200 MHz, δ, ppm): 3.43 (t, 2H, CH₂N, J=8 Hz), 3.06 (bs,2H), 2.15-0.70 (m, 11H), 1.43 (s, 18H, tert-butyl).

Step C.14-O-{[(1R,2R)-5-(3-N,N-Bis-(tert-butoxycarbonyl)-amino-propyl)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer and14-O-{[(1R,2R)-4-(3-N,N-Bis-(tert-butoxycarbonyl)-amino-propyl)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer

N-tert-Butoxycarbonyl-[3-(7-oxa-bicyclo[4.1.0]hept-3-yl)-propyl]carbamicacid tert-butyl ester (3.00 g, 8.44 mmol) was treated with pleuromutilinthiol (5.00 g, 12.7 mmol) according to the method of Example 1 Step A3and stirred for 24 h. After chromatography of the mixture (silica,petroleum benzene/ethyl acetate=7/3→1/1) a mixture of the titlecompounds (petroleum benzene/ethyl acetate=3/2, R₁=0.30, 3.68 g, 58%yield, uncorrected) was obtained as white foam.

¹H NMR (DMSO-d₆, 500 MHz, δ, ppm, inter alia): 6.12 (dd, 1H, 19-H, J=11Hz and 18 Hz), 5.53 (d, 1H, 14-H, J=8 Hz), 5.10-5.00 (m, 2H, 20-H), 4.75(bs, 1H), 4.48 (bs, 1H, 11-OH), 3.69 (m, 0.5H), 3.60 (m, 0.5H),3.45-3.38 (m, 3H), 3.26 (d, 1H, 22-H, J=12 Hz), 3.21 (d, 1H, 22-H, J=13Hz), 2.91 (m, 0.5H), 2.85 (m, 0.5H), 2.39 (bs, 1H, 4-H), 2.18 (dd, 1H,2-H, J=11 Hz and 19 Hz), 2.12-2.01 (m, 3H), 1.92 (m, 1H), 1.70-0.96 (m),1.41 (s, 18H, tert-butyl), 1.35 (s, 3H, 15-CH3), 1.05 (s, 3H, 18-CH₃),0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.62 (d, 3H, 16-CH₃, J=7 Hz).

Step D.14-O-{[(1R,2R)-5-(3-Amino-propyl)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer and14-O-{[(1R,2R)-4-(3-Amino-propyl)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S)diastereomer

A mixture of the mixture from Example 33 Step C (250 mg, 0.33 mmol) wastreated with trifluoroacetic acid (6 ml) according to the method ofExample 1 Step B, and stirred at room temperature for 5 days. After workup and chromatography (silica, dichloromethane/methanol/28% aqNH₄OH=86:10:4) a mixture of the title compounds (R_(f)=0.08 and 0.12, 50mg, 27% yield, uncorrected) was obtained as white foam.

¹H NMR (DMSO-d₆, 500 MHz, δ, ppm, inter alia): 6.14 (dd, 1H, 19-H,J=11Hz and 18 Hz), 5.55 (d, 1H, 14-H, J=8 Hz), 5.13-5.02 (m, 2H, 20-H),4.75 (bs, 1H), 4.50 (bs, 1H, 11-OH), 3.72 (m, 0.5H), 3.61 (m, 0.5H),3.50-3.05 (m), 2.92 (m, 0.5H), 2.86 (m, 0.5H), 2.47 (m, 2H, CH₂N), 2.41(bs, 1H, 4-H), 2.19 (dd, 1H, 2-H), 2.13-2.03 (m, 3H), 1.94 (m, 1H),1.73-0.82 (m), 1.37 (s, 3H, 15-CH3), 1.06 (s, 3H, 18-CH₃), 0.82 (d, 3H,17-CH₃, J=7 Hz), 0.63 (d, J=7 Hz, 3H, 16-CH₃). MS-ESI (m/z): 550 (MH⁺),572 (MNa⁺).

Example 34. 14-O-[(4-Amino-2-oxo-cyclohexylsulfanyl)-acetyl]-mutilinStep A. Thiobenzoic acidS-((1R,2R,4R)-4-tert-butoxycarbonylamino-2-hydroxy-cyclohexyl)ester+(1S,2S,4S) diastereomer and Thiobenzoic acidS-((1R,2R,5S)-5-tert-butoxycarbonylamino-2-hydroxy-cyclohexyl)ester+(1S,2S,5R) diastereomer

To a solution of syn-3,4-epoxycyclohexyl-carbamic acid tert-butyl ester(63.3 g, 0.29 mol) in 630 ml of toluene was added thiobenzoic acid(105.13 ml, 0.90 mmol) followed by tetrabutyl ammonium chloridemonohydrate (2.66 g, 9.00 mmol). The mixture was stirred under argon for3.5 hours and was charged with saturated aqueous sodium bicarbonate,stirred for 10 min and the organic phase was separated. The organicphase was washed with saturated aqueous sodium bicarbonate, brine, driedand the solvent was removed under vacuum to obtain the crude mixture ofthe title compounds. The mixture was crystallized from a mixture oftoluene/hepane (1/1) to give thiobenzoic acidS-((1R,2R,4R)-4-tert-butoxycarbonylamino-2-hydroxy-cyclohexyl)ester+(1S,2S,4S) diastereomer (a) (22.1 g, 21% yield) as a solid. Themother liquor was chromatographed (silica, toluol/ethylacetate=8/1−>7/1) to obtain thiobenzoic acidS-((1R,2R,4R)-4-tert-butoxycarbonylamino-2-hydroxy-cyclohexyl)ester+(1S,2S,4S) diastereomer (a) (toluene/ethyl acetate=3/1,R_(f)=0.35, 4.09 g, 4% yield) as a solid and thiobenzoic acidS-((1R,2R,5S)-5-tert-butoxycarbonylamino-2-hydroxy-cyclohexyl)ester+(1S,2S,5R) diastereomer (b) (toluene/ethyl acetate=3/1,R_(f)=0.25, 16.57 g, 16% yield) as oil.

(a): ¹H NMR (500 MHz, DMSO-d₆, δ, ppm): 7.88 (d, 2H, aromatic H, J=7Hz), 7.66 (t, 1H, aromatic H, J=7 Hz), 7.53 (t, 2H, aromatic H, J=8 Hz),6.82 (d, 1H, NH, J=8 Hz), 5.08 (d, 1H, OH, J=6 Hz), 3.41 (m, 1H),3.33-3.28 (m, 2H), 2.08 (bd, 1H), 1.98 (m, 1H), 1.75 (m, 1H), 1.36 (s,9H, tert-butyl), 1.49-1.18 (m, 3H).

(b): ¹H NMR (500 MHz, DMSO-d₆, δ, ppm): 7.91 (d, 2H, aromatic H, J=8Hz), 7.67 (t, 1H, aromatic H, J=7 Hz), 7.53 (t, 2H, aromatic H, J=8 Hz),6.85 (d, 1H, NH, J=7 Hz), 5.12 (d, 1H, OH, J=3 Hz), 3.89 (d, 1H, J=4Hz), 3.62 (bs, 1H), 3.41 (bs, 1H), 2.10 (m, 1H), 1.69-1.48 (m, 5H), 1.35(s, 9H, tert-butyl).

Step B. Thiobenzoic acid S-(4-tert-butoxycarbonylamino-2-oxo-cyclohexyl)ester

A mixture of thiobenzoic acidS-((1R,2R,4R)-4-tert-butoxycarbonylamino-2-hydroxy-cyclohexyl)ester+(1S,2S,4S) diastereomer (5 g, 14.2 mmol), 4 Å-molecular sieve (3g) 1,1-dihydro-1,1,1-triacetoxy-1,2-benziodoxol-3(1H)-one (6.33 g, 15mmol) in 100 ml of dichloromethane was stirred under argon at 5° C. for1 hour and 1 hour at room temperature. The mixture was filtered overcelite, dried and concentrated under vacuum to give the crude product.After chromatography of the mixture (silica, toluene/ethyl acetate=7/1)the title compound (toluene/ethyl acetate=3/1, R_(f)=0.48, 4.18 g, 84%yield) was obtained as a white solid.

¹H NMR (DMSO-d₆, 200 MHz, δ, ppm): 7.83 (d, 2H, aromatic H, J=7 Hz,),7.71 (t, 1H, aromatic H, J=7 Hz), 7.57 (t, 2H, aromatic H, J=7 Hz), 7.16(bd, 1H, NH, J=8 Hz), 4.48 (m, 1H), 3.98, 3.7 (2m, 1H), 2.58 (m, 2H),2.31-1.68 (m, 4H), 1.39 (s, 9H, tert-butyl).

Step C.14-O-[(2-Benzoyloxy-4-tert-butxycarbonylamino-cyclohex-1-en-ylsulfanyl)-acetyl]-mutilin

A mixture of thiobenzoic acidS-(4-tert-butoxycarbonylamino-2-oxo-cyclohexyl) ester (2 g, 5.72 mmol),pleuromutilin tosylate (3.96 g, 7.44 mmol), potassium carbonate (1.58 g,11.44 mmol) and tetrabutyl ammonium chloride monohydrate (0.2 g, 0.68mmol) in 20 ml of dimethyl formamide and 2 ml of water was stirred for24 hours. The mixture was taken up in ethyl acetate, washed with aqueoussaturated sodium bicarbonate and brine, dried and concentrated undervacuum. After chromatography of the mixture (silica, toluene/ethylacetate=5/1) the title compound (toluene/ethyl acetate=3/1, R_(f)=0.24,2.36 g, 58% yield) was obtained as white foam.

¹H NMR (DMSO-d₆, 400 MHz, δ, ppm, inter alia): 7.99 (d, 2H, aromatic H,J=7 Hz), 730 (t, 1H, aromatic H, J=7 Hz), 7.54 (t, 2H, aromatic H, J=8Hz), 6.91 (bd, 1H, NH, J=5 Hz), 6.13 (dd, 1H, 19-H, J=18 Hz and 11 Hz),5.53 (dd, 1H, 14-H, J=3 Hz and 8 Hz), 5.10 (dd, 1H, 20-H, J=2 Hz and 17Hz), 5.04 (dd, 1H, 20-H, J=2 Hz and 11 Hz), 4.49 (d, 1H, 11-OH, J=6 Hz),3.64 (bs, 4′-H, 1H), 3.49-3.35 (m, 3H), 2.46-2.34 (m, 3H), 2.28-2.02 (m,4H), 1.87 (m, 1H), 1.67-1.42 (m, 5H), 1.41-1.36 (m, 1H), 1.37 (s, 9H,tert-butyl), 1.34 (s, 3H, 15-CH₃), 1.23 (m, 1H), 1.06 (s, 3H, 18-CH₃),1.00 (m, 1H), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.58 (d, 3H, 16-CH₃, J=7 Hz,3H). MS-ESI (m/z): 732 (MNa⁺).

Step D.14-O-[(4-tert-Butoxycarbonylamino-2-oxo-cyclohexylsulfanyl)-acetyl]-mutilin

A solution of14-O-[(2-benzoyloxy-4-tert-butoxycarbonylamino-cyclohex-1-en-ylsulfanyl)-acetyl]-mutilin(0.8 g, 1.13 mmol) in 8 ml of methanol and 1.25 ml of aqueous 1M sodiumhydroxide (1.25 mmol) was stirred for 0.5 hours. The mixture was dilutedwith water and extracted with dichloromethane. The organic extract wasdried over magnesium sulfate and concentrated under vacuum to give amixture. After chromatography of the mixture (silica, toluene/ethylacetate=5/1) the title compound (toluene/ethyl acetate=3/1, R_(f)=0.13,0.57 g, 83% yield) was obtained as white foam.

¹H NMR (DMSO-d₆, 400 MHz, δ, ppm, inter alia): 7.04 (m, 1H, NH), 6.12(m, 1H, 19-H), 5.53 (m, 1H, 14-H), 5.09-5.02 (m, 2H, 20-H), 4.49 (m, 1H,11-OH), 3.65 (m, 1H, 4′-H), 3.40 (m, 1H), 3.32-3.14 (m, 2H), 2.39 (s,1H, 4-H), 2.40-0.95 (m), 1.35 (s, 9H, tert-butyl), 1.34 (s, 3H, 15-CH₃),1.05 (s, 3H, 18-CH₃), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.60 (d, 3H, 16-CH₃,J=7 Hz). MS-ESI (m/z): 628 (MNa⁺).

Step E. 14-O-[(4-Amino-2-oxo-cyclohexylsulfanyl)-acetyl]-mutilin

14-O-[(4-tert-Butoxycarbonylamino-2-oxo-cyclohexylsulfanyl)-acetyl]-mutilin(0.55 g, 0.91 mmol) was treated with 2.3 ml of 4M HCl in dioxane (9.2mmol) according to the method of Example 32 Step F and stirred for 2hours at room temperature. After work up the title compound (0.42 g, 91%yield) was obtained as white foam.

¹H NMR (DMSO-d₆, 400 MHz, δ, ppm, inter alia): 6.13 (m, 1H, 19-H), 5.53(d, 1H, 14-H, J=4 Hz), 5.05 (m, 2H, 20-H), 4.48 (bs, 1H, 11-OH),3.55-3.10 (m), 2.41-2.30 (m), 2.22-2.13 (m, 2H), 2.12-1.99 (m, 3H),1.69-1.55 (m, 3H), 1.55-1.20 (m), 1.35 (s, 3H, 15-CH₃), 1.05 (s, 3H,18-CH₃), 0.99 (m, 1H), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.61 (d, 3H,16-CH₃).

Example 35—14-O-[(5-Amino-2-oxo-cyclohexylsulfanyl)-acetyl]-mutilinsuccinic acid salt Step A. Thiobenzoic acidS-(5-tert-butoxycarbonylamino-2-oxo-cyclohexyl) ester

Thiobenzoic acidS-((1R,2R,5S)-5-tert-butoxycarbonylamino-2-hydroxy-cyclohexyl)ester+(1S,2S,5R) diastereomer (8.7 g, 24.8 mmol) from Example 34 Step Awas treated with 1,1-dihydro-1,1,1-triacetoxy-1,2-benziodoxol-3(1H)-one(11.02 g, 26 mmol) according to the method of Example 34 Step B. Afterwork up and chromatography (silica, toluene/ethyl acetate=6/1) of themixture the title compound (toluene/ethyl acetate=3/1, R_(f)=0.43, 7.15g, 83% yield) was obtained as a white solid.

¹H NMR (DMSO-d₆, 200 MHz, δ, ppm): 7.91 (d, 2H, aromatic H, J=7 Hz),7.71 (t, 1H, aromatic H, J=7 Hz), 7.55 (t, 2H, aromatic H, J=7 Hz), 6.97(d, 1H, NH, J=7 Hz), 4.70 (m, 1H), 4.02 (m, 1H), 2.79 (m, 1H), 2.46-2.22(m, 2H), 2.18-1.93 (m, 2H), 1.75 (m, 1H), 1.39 (s, 9H, tert-butyl).

Step B.14-O-[(2-Benzoyloxy-5-tert-butoxycarbonylamino-cyclohex-1-en-ylsulfanyl)-acetyl]-mutilin

Thiobenzoic acid S-(5-tert-butoxycarbonylamino-2-oxo-cyclohexyl) ester(1 g, 2.86 mmol) was treated with pleuromutilin tosylate (1.98 g, 3.72mmol) according to the method of Example 34 Step C. After work up andchromatography (silica, toluene/ethyl acetate=5/1) of the mixture thetitle compound (toluene/ethyl acetate=3/1, R_(f)=0.23, 1.33 g, 65%yield) was obtained as white foam.

¹H NMR (DMSO-d₆, 500 MHz, δ, ppm, inter alia): 8.00 (d, 2H, aromatic H,J=8 Hz), 7.70 (t, 1H, aromatic H, J=7 Hz), 7.54 (t, 2H, aromatic H, J=8Hz), 6.96 (d, 0.5 H, J=8 Hz), 6.92 (d, 0.5H, J=8 Hz), 6.12 (dd, 1H,19-H, J=18 Hz and 11 Hz), 5.52 (dd, 1H, 14-H, J=3 Hz and 8 Hz), 5.08(dd, 1H, H-20, J=2 Hz and 18 Hz), 5.05 (dd, 1H, H-20, J=2 Hz and 11 Hz),4.51 (t or 2d, 1H, J=6 Hz), 3.63 (bs, 1H), 3.47-3.33 (m, 3H), 2.55 (m,1H), 2.44-2.32 (m, 2H), 2.29-2.14 (m, 3H), 2.10-2.01 (m, 3H), 1.84 (m,1H), 1.68-1.57 (m, 3H), 1.46 (m, 1H), 1.39 (s, 9H, tert-butyl), 1.34,1.32 (2s, 3H, 15-CH₃), 1.29-1.18 (m, 3H), 1.05 (s, 3H, 18-CH₃), 0.98 (m,1H), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.58 (d, 3H, 16-CH₃, J=7 Hz). MS-ESI(m/z): 732 (MNa⁺).

Step C.14-O-[(5-tert-Butoxycarbonylamino-2-ozo-cyclohexylsulfanyl)-acetyl]-mutilin

14-O-[(2-Benzoyloxy-5-tert-butoxycarbonylamino-cyclohex-1-en-ylsulfanyl)-acetyl]-mutilin(1 g, 1.41 mmol) was treated with 1.55 ml of aqueous 1M sodium hydroxide(1.55 mmol) according to Example 34 Step D. After work up andchromatography of the mixture (silica, toluene/ethyl acetate=5/1) thetitle compound (toluene/ethyl acetate=3/1, R_(f)=0.22, 0.6 g, 70% yield)was obtained as white foam.

¹H NMR (DMSO-d₆, 400 MHz, δ, ppm, inter alia): 6.89 (d, 0.5H, NH, J=6Hz), 6.86 (d, 0.5H, J=8 Hz), 6.17-6.05 (m, 1H, 19-H), 5.51 (d, 1H, 14-H,J=8 Hz), 5.09-4.99 (m, 2H, 20-H), 4.47 (m, 1H, 11-OH), 3.92, 3.55 (2m,1H, 1′-H), 3.79 (m, 1H, 5′-H), 3.40 (t, 1H, 11-H, J=6 Hz), 3.35-3.12 (m,2H), 2.77, 2.50 (2m, 1H), 2.41-1.90 (m), 1.71-1.18 (m), 1.37 (s, 9H,tert-butyl), 1.35 (s, 3H, 15-CH₃), 1.04 (s, 3H, 18-CH₃), 0.99 (m, 1H),0.80 (d, 3H, 17-CH₃, J=7 Hz), 0.59 (d, 3H, 16-CH₃, J=6 Hz).

Step D. 14-O-[(5-Amino-2-oxo-cyclohexylsulfanyl)-acetyl]-mutilin

14-O-[(5-tert-Butoxycarbonylamino-2-oxo-cyclohexylsulfanyl)-acetyl]-mutilin(0.3 g, 0.5 mmol) was treated with 1.25 ml of 4M HCl in dioxane (5 mmol)according to the method of Example 32 Step F and stirred for 2 hours.After work up the title compound (0.26 g, quantitative yield,uncorrected) was obtained as white foam and was directly taken to thenext step.

MS-ESI (m/z): 506.0 (MH⁺).

Step E. 14-O-[(5-Amino-2-oxo-cyclohexylsulfanyl)-acetyl]-mutilinsuccinic acid salt

A solution of succinic acid (59 mg, 0.5 mmol) in 5 ml of iso-propanolwas added under stirring over a period of 5 min to a solution of14-O-[(5-Amino-2-oxo-cyclohexylsulfanyl)-acetyl]-mutilin (0.26 g, 0.5mmol) in 10 ml of methyl tert-butyl ether and 5 ml of isopropanol. Themixture was stirred for 4 h and stripped of the solvent. The residue wasdissolved in 2 ml of isopropanol and 20 ml of methyl tert-butyl etherwere added to it and stirred for 1 hour. The precipitate was filtered,washed with 5 ml of methyl tert-butyl ether and dried under vacuum toobtain the title compound (0.18 g, 58% yield) as a white solid.

¹H NMR (DMSO-d₆, 400 MHz, δ, ppm, inter alia): 6.18-6.05 (m, 1H, H-19),5.52 (d, 1H, 14-H, J=7 Hz), 5.12-4.98 (m, 2H, 20-H), 4.50 (bs, 1H,11-OH), 3.89, 3.53 (2m, 1 H, 1′-H), 3.42-3.14 (m), 2.39 (s, 1H), 2.27(s, 2H), 2.23-1.96 (m), 1.69-1.17 (m), 1.34 (s, 3H, 15-CH₃), 1.08 (s,3H, 18-CH₃), 1.13-0.93 (m), 0.80 (d, 3H, 17-CH₃, J=7 Hz), 0.59 (d, 3H,16-CH₃, J=6 Hz).

Example36—14-O-{[(6R,8R)-8-Amino-1,4-dioxa-spiro[4.5]dec-6-ylsulfanyl]-acetyl}-mutilinsuccinic acid salt+(6S,8S) diastereomer succinic acid salt Step A.Thiobenzoic acidS-((6R,8R)-8-tert-butoxycarbonylamino-1,4-dioxa-spiro[4.5]dec-6-yl)ester+(65, 8S) diastereomer

A mixture of the compound of Example 35 Step A (2.00 g, 5.72 mmol),1,2-ethandiol (2.0 ml, 35.8 mmol), 25 ml of dichloromethane and BF₃ (48%purity, 0.60 ml, 4.25 mmol) was stirred for 2 days. The mixture wasdiluted with dichloromethane and washed with saturated aqueous sodiumbicarbonate and brine, dried and stripped of the solvent. Afterchromatography (silica, ethyl acetate/cyclohexane=1/9−>15/85) the titlecompound (toluene/ethyl acetate=9/1, R_(f)=0.28, 632 mg, 28% yield) wasobtained as foam.

¹H NMR (DMSO-d₆, 400 MHz, δ, ppm): 7.89 (m, 2H, aromatic H), 7.66 (t,1H, aromatic H, J=8 Hz), 7.53 (t, 2H, aromatic H, J=8 Hz), 6.86 (d, 1H,NH, J=8 Hz), 4.04-3.83 (m, 5H), 3.48 (m, 1H), 2.09-1.40 (m, 6H), 1.35(s, 9H, tert-butyl).

Step B. ((6R,8R)-6-Mercapto-1,4-dioxa-spiro[4.5]dec-8-yl)-carbamic acidtert-butyl ester+(6S,8S) diastereomer

A mixture of thiobenzoic acidS-((6R,8R)-8-tert-butoxycarbonylamino-1,4-dioxa-spiro[4.5]dec-6-yl)ester+(6S,8S) diastereomer (695 mg, 1.77 mmol) and hydrazine (80% aq.solution, 0.10 ml, 2.65 mmol) in 7 ml of dichloromethane was stirred for24 hours. The mixture was diluted with dichloromethane and washed with1M aqueous HCl, dried and stripped of the solvent to obtain the titlecompound (490 mg, 95% yield) as yellow-gray oil.

¹H NMR (CDCl₃, 400 MHz, δ, ppm, inter alia): 4.34 (bs, 1H, NH),4.13-4.05 (m, 2H), 3.95-3.85 (m, 2H), 3.49 (bs, 1H), 2.98 (m, 1H), 2.25(m, 1H), 1.90-1.75 (m, 2H), 1.69-1.41 (m), 1.37 (s, 9H, tert-butyl).

Step C.14-O-{[(6R,8R)-8-tert-Butoxycarbonylamino-1,4-dioxa-spiro[4.5]dec-6-ylsulfanyl]-acetyl}-mutilin+(6S,8S)diastereomer

((6R,8R)-6-Mercapto-1,4-dioxa-spiro[4.5]dec-8-yl)-carbamic acidtert-butyl ester+(6S,8S) diastereomer (410 mg, 1.42 mmol) was treatedwith pleuromutilin tosylate (910 mg, 1.71 mmol) according to the methodof Example 1 Step A3 and stirred for 2.5 hours. The mixture wasconcentrated in vacuo and the residue was taken up in ethyl acetate,washed with water, 0.1M aqueous HCl, aqueous sodium bicarbonate andbrine, dried and stripped of the solvent. After chromatography (silica,ethyl acetate/toluene=1/4) of the mixture the title compound(R_(f)=0.18, 613 mg, 66% yield) was obtained as pale yellow foam.

¹H NMR (DMSO-d₆, 400 MHz, δ, ppm, inter alia): 6.77 (bm, 1H, NH), 6.13(dd, 1H, 19-H, J=11 Hz and 18 Hz), 5.54 (d, 1H, 14-H, J=8 Hz), 5.11-5.02(m, 2H, H-20), 4.47 (m, 1H, 11-OH), 4.04-3.81 (m, 4H), 3.39 (m, 1H,11-H), 3.35-3.17 (m, 3H), 3.02 (m, 1H), 2.39 (s, 1H, 4-H), 2.25-1.93(m), 1.75-1.18 (m), 1.36 (2s, 12H, 15-CH₃, tert-butyl), 1.05-0.95 (m),1.05 (s, 3H, 18-CH₃), 0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.62 (d, 3H, 16-CH₃,J=7 Hz). MS-ESI (m/z): 672 (MNa⁺).

Step D.14-O-{[(6R,8R)-8-Amino-1,4-dioxa-spiro[4.5]dec-6-ylsulfanyl]-acetyl}-mutilin+(6S,8S)diastereomer

14-O-{[(6R,8R)-8-tert-Butoxycarbonylamino-1,4-dioxa-spiro[4.5]dec-6-ylsulfanyl]-acetyl}-mutilin+(6S,8S)diastereomer (250 mg, 0.39 mmol) was treated with 1.5 ml trifluoroaceticacid according to the method of Example 1 Step B and stirred for 6 hoursat room temperature. After work up and chromatography (silica,dichloromethane/methanol=19/1) the title compound(dichloromethane/methanol=9/1, R_(f)=0.15-0.54, 160 mg, 76% yield) wasobtained as white foam, which was directly taken to the next step.

Step E.14-O-{[(6R,8R)-8-Amino-1,4-dioxa-spiro[4.5]dec-6-ylsulfanyl]-acetyl}-mutilinsuccinic acid salt+(6S,8S) diastereomer succinic acid salt

14-O-{[(6R,8R)-8-Amino-1,4-dioxa-spiro[4.5]dec-6-ylsulfanyl]-acetyl}-mutilin(6S,8S) diastereomer (120 mg, 0.22 mmol) was treated with succinic acid(25.7 mg, 0.22 mmol) according to the method of Example 35 Step E toobtain the title compound (100 mg, 69% yield) as a pale yellow solid.

¹H NMR (DMSO-d₆, 400 MHz, δ, ppm, inter alia): 6.16, 6.15 (2dd, 1H,19-H, J=11 Hz and 18 Hz), 5.56 (d, 1H, 14-H, J=8 Hz), 5.14-5.02 (m, 2H,20-H), 4.53 (bs), 4.08-3.85 (m), 3.48-3.21 (m), 3.12-3.01 (m), 2.42 (bs,1H, 4-H), 2.30 (s, 4H, succinic acid), 2.26-2.02 (m), 1.84-1.21 (m),1.38 (s, 3H, 15-CH₃), 1.08 (s, 3H, 18-CH₃), 0.83 (d, 3H, 17-CH₃, J=7Hz), 0.64 (d, 3H, 16-CH₃, J=7 Hz).

Example 37—14-O-{[5-Amino-2-methoxy-cyclohexylsulfanyl]-acetyl}-mutilinand 14-O-{[4-Amino-2-methoxy-cyclohexylsulfanyl]-acetyl}-mutilin Step A.14-O-{[5-tert-Butoxycarbonylamino-2-methoxy-cyclohexylsulfanyl]-acetyl}-mutilinand14-O-{[4-tert-Butoxycarbonylamino-2-methoxy-cyclohexylsulfanyl]-acetyl}-mutilin

An ice-cold solution of14-O-{[(1R,2R,5S)-5-tert-butoxycarbonylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin+(1S,2S,5R)diastereomer (4.00 g, 6.58 mmol) from Example 1 Step A in 120 ml ofdichloromethane was treated with BF₃ (48% purity, 0.20 ml, 1.65 mmol)followed by trimethylsilyl diazomethane (2M in hexane, 0.82 ml, 1.64mmol) under stirring. After 20 minutes 3 additional amounts of BF₃ (48%purity, 0.20 ml, 1.65 mmol) followed by 3 additional amounts oftrimethylsilyl diazomethane (2M in hexane, 0.82 ml, 1.64 mmol) wereadded each time in time interval of 20 min. The mixture was stirred atroom temperature for 30 min, charged with 200 ml of saturated aqueoussodium bicarbonate and stirred. The organic phase was separated andwashed with saturated aqueous sodium bicarbonate, dried and stripped ofthe solvent to give a mixture. After chromatography (silica, ethylacetate/cyclohexane=1/4−>1/1) a mixture of the title compounds (0.49 g,12% yield) was obtained as a mixture as white foam.

¹H NMR (DMSO-d₆, 500 MHz, δ, ppm, inter alia): 6.72 (bm, 1H, NH),6.17-6.09 (2dd, 1H, 19-H, J=11 Hz and 18 Hz), 5.54 (2d, 1H, 14-H, J=8Hz), 5.10-5.00 (m, 2H, 20-H), 4.49 (d, 11-OH, J=6 Hz), 3.50-3.17 (m),3.21 (2s, 3H, OMe), 2.40 (bs, 1H, 4-H), 2.26-2.01 (m), 1.73-1.56 (m),1.53-0.96 (m), 1.35 (2s, 12H, 15-CH₃, tert-butyl), 1.05 (s, 3H, 18-CH₃),0.81 (d, 3H, 17-CH₃, J=7 Hz), 0.62 (d, 3H, 16-CH₃, J=7 Hz).

Step B.14-O-{[5-tert-Butoxycarbonylamino-2-methoxy-cyclohexylsulfanyl]-acetyl}-mutilinand14-O-{[4-tert-Butoxycarbonylamino-2-methoxy-cyclohexylsulfanyl]-acetyl}-mutilin

14-O-{[5-tert-Butoxycarbonylamino-2-methoxy-cyclohexylsulfanyl]-acetyl}-mutilinand14-O-{[4-tert-Butoxycarbonylamino-2-methoxy-cyclohexylsulfanyl]-acetyl}-mutilin(80 mg, 0.13 mmol) was treated with 0.32 ml of 4M HCl in dioxane (1.28mmol) according to the method of Example 32 Step F and stirred for 2hours. After work up and chromatography (silica, ethylacetate/methanol/28% aq NH₃=500/100/1) a mixture of the title compounds(12 mg, 18% yield, uncorrected) was obtained as white foam.

¹H NMR (DMSO-d₆, 500 MHz, δ, ppm, inter alia): 6.18-6.05 (m, 1H, 19H),5.55 (m, 1H, 14-H), 5.10-5.00 (m, 2H, 20-H), 4.58, 4.50 (2m, 1H, 11-OH),3.55-3.20 (m), 2.86-2.72 (m), 2.44, 2.40 (2s, 1H, 4-H), 2.22-1.99 (m),1.78-1.20 (m), 1.06, 1.05 (2s, 3H, 18-CH₃), 1.03 (m, 1H), 0.81 (m, 3H,17-CH₃), 0.62 (m, 3H, 18-CH₃). MS-ESI (m/z): 522 (MF⁺).

1. A compound of formula (I)

wherein n is 0 to 4; m is 0 or 1 with the proviso that the sulphur atomand R₃ are in vicinal position (if m=0 then R₃ is in position 2′, and ifm=1 then R₃ is on position 1′); R is ethyl or vinyl; R₁ is hydrogen or(C₁₋₆)alkyl, R₂ is hydrogen or (C₃₋₆)cycloalkyl, or unsubstituted(C₁₋₆)alkyl, or (C₁₋₆)alkyl substituted by one or more of hydroxy;preferably one or two, methoxy, halogen, (C₃₋₆)cycloalkyl, or R₁ and R₂together with the nitrogen atom to which they are attached form a 5 to 7membered heterocyclic ring containing at least 1 nitrogen atom or 1nitrogen and 1 additional heteroatoms e.g. selected from N or O, or R₁is hydroxy and R₂ is formyl; R₃ is OH, OR₄, a halogen atom, or with theproviso that R₃ is bound to 2′ R₃ represents —O—(CH₂)_(p)—O— with p is 2or 3; R₄ is unsubstituted (C₁₋₆)alkyl or (C₃₋₆)cycloalkyl.
 2. A compoundof formula (II)

wherein n, R, R₁, R₂ and R₃ are as defined in claim
 1. 3. A compound offormula (III)

wherein n, R, R₁ and R₂ are as defined in claim
 1. 4. A compound offormula (IV)

wherein n, R₁ and R₂ are as defined in claim
 1. 5. A compound of formula(V)

wherein n, R₁ and R₂ are as defined in claim
 1. 6. A compound of formula(VI)

wherein n, R₁ and R₂ are as defined in claim
 1. 7. A compound accordingto one of the claims 1 to 6, selected from the group consisting of14-O-{[(1R,2R,4R)-4-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin14-O-{[(1S,2S,4S)-4-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin14-O-{[(1R,2R,5S)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin14-O-{[(1S,2S,5R)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin14-O-{[(1R,2R,4S)-4-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S,4R) diastereomer thereof14-O-{[(1R,2R,5R)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin14-O-{[(1S,2S,5S)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilin14-O-{[(1R,2R,3R)-3-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S,3S) diastereomer thereof14-O-{[(1R,2R,4R)-4-Diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S,4S) diastereomer thereof14-O-{[(1R,2R,4R)-4-Ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S,4S) diastereomer thereof14-O-{[(1R,2R,5S)-5-Ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S,5R) diastereomer thereof14-O-{[(1R,2R,5S)-5-Diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S,5R) diastereomer thereof14-O-{[(1R,2R,4S)-4-Diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S,4R) diastereomer thereof14-O-{[(1R,2R,5R)-5-Diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S,5S) diastereomer thereof14-O-{[(1R,2R,3R)-3-Ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S,3S) diastereomer thereof14-O-{[(1R,2R,3R)-3-Diethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S,3S) diastereomer thereof14-O-{[(1R,2R,4S)-4-(Formyl-hydroxy-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S,4R) diastereomer thereof14-O-{[(1R,2R,5S)-5-(Formyl-hydroxy-amino)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S,5R) diastereomer thereof14-O-{[(1R,2R,3R/S)-3-(Formyl-hydroxy-amino)-2-hydroxy-cyclohexylsulfatyl]-acetyl}-mutilinand the (1S,2S,3R/S) diastereomer thereof14-O-{[(1R,2R,5S)-2-Hydroxy-5-methylamino-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S,5R) diastereomer thereof14-O-{[(1R,2R,5S)-5-Allylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S,5R) diastereomer thereof14-O-{[(1R,2R,5S)-2-Hydroxy-5-(2-methoxy-ethylamino)-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S,5R) diastereomer thereof14-O-{[(1R,2R,4R*)-2-Hydroxy-4-(2-hydroxy-ethylamino)-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S,4S*) diastereomer thereof14-O-{[(1R,2R,4R*)-4-Cyclohexylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S,4S*) diastereomer thereof14-O-{[(1R,2R,4R*)-4-Cyclopropylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S,4S*) diastereomer thereof14-O-{[(1R,2R,5S*)-4-Cyclopropylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S,5R*) diastereomer thereof14-O-{[(1R,2R,4S*)-4-Cyclopropylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S,4R*) diastereomer thereof14-O-{[(1R,2R,5R*)-2-Hydroxy-5-morpholin-4-yl-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S,5S*) diastereomer thereof14-O-{[(1R,2R,5S*)-2-Hydroxy-5-morpholin-4-yl-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S,5R*) diastereomer thereof14-O-{[(1R,2R,5S)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilinand the (1S,2S,5R) diastereomer thereof14-O-{[(1R,2R,5S)-5-Ethylamino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilinand the (1 S,2S,5R) diastereomer thereof14-O-{[(1R,2R,5R)-5-Amino-2-hydroxy-cyclohexylsulfanyl]-acetyl}-19,20-dihydro-mutilinand the (1 S,2S,5S) diastereomer thereof14-O-{[(1R,2R)-4-Aminomethyl-2-hydroxy-cyclohexylsufanyl]-acetyl}-mutilinand the (1S,2S) diastereomers thereof14-O-{[5-Amino-2-chloro-cyclohexylsulfanyl]-acetyl}-mutilin14-O-{[4-Amino-2-chloro-cyclohexylsulfanyl]-acetyl}-mutilin14-O-[(4-Amino-1-hydroxy-cyclohexylmethylsulfanyl)-acetyl]-mutilin14-O-{[(1R,2R)-2-Hydroxy-5-(3-methylamino-propyl)-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S) diastereomers thereof14-O-{[(1R,2R)-2-Hydroxy-4-(3-methylamino-propyl)-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S) diastereomers thereof14-O-{[(1R,2R)-5-(3-Amino-propyl)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S) diastereomers thereof14-O-{[(1R,2R)-4-(3-Amino-propyl)-2-hydroxy-cyclohexylsulfanyl]-acetyl}-mutilinand the (1S,2S) diastereomer thereof14-O-{[(6R,8R)-8-Amino-1,4-dioxa-spiro[4.5]dec-6-ylsulfanyl]-acetyl}-mutilinand the (6S,8S) diastereomer thereof14-O-{[4-Amino-2-methoxy-cyclohexylsulfanyl]-acetyl}-mutilin and14-O-{[5-Amino-2-methoxy-cyclohexylsulfanyl]-acetyl}-mutilin
 8. Thecompound according to any of claims 1 to 7 in the form of a salt and/orsolvate.
 9. A compound according to any of claims 1 to 8 for use as apharmaceutical drug substance.
 10. A method of treatment of diseasesmediated by microbes which comprises administering to a subject in needof such treatment an effective amount of a compound of any one of claims1 to
 8. 11. A pharmaceutical drug composition comprising a compound ofanyone of claims 1 to 8, in association with at least one pharmaceuticalexcipient.
 12. A pharmaceutical drug composition according to claim 11,further comprising another pharmaceutically active agent.